Introduction: Chronic spontaneous urticaria (CSU) is defined as the presence of hives, angioedema, or both, accompanied by intense itch, in a duration longer than 6 weeks. CSU affects about 1% of the general population and there has been a noticeable increase in the prevalence of this condition in recent years. It is more frequently observed in young and middle-aged adults, affecting women two times more often than men. The average duration of CSU is commonly around 5 years, although, in 10–25% of patients, it persists for more than 5 years. The treatment of CSU is focused on “symptom control”, targeting mast cell mediators and activators such as histamine and autoantibodies. Second-generation non-sedating H1 antihistamines are recommended as the initial treatment (up to four-fold dose), followed by omalizumab as a second-line treatment. Other therapies include cyclosporin, glucocorticoids, and alternative treatments with limited evidence of efficacy. Debilitating symptoms and the prolonged duration of the disease significantly impact patients` quality of life. Although the precise etiopathogenesis is largely unknown, CSU is considered an immune-mediated inflammatory disease, with an autoimmune etiology in more than 50% of cases. To date, 2 autoimmune endotypes are recognized. Type I autoimmune (autoallergic) CSU is characterized by the presence of IgE antibodies against autoantigens, for example, thyroid peroxidase and IL-24. Type IIb autoimmune CSU is associated with IgG autoantibodies to IgE and or its highaffinity receptors on mast cells (FcεRI). Human microbiota plays a crucial role in the development and maintenance of immune system balance. Gut and salivary microbiota are two major communities of microorganisms within the human body, including bacteria, fungi, and viruses. They play an important role in health maintenance, such as preventing pathogen propagation, controlling metabolism, improving epithelial integrity, and regulating the components of both innate and adaptive host immune systems. Smoking, alcohol consumption, overuse of antibiotics, poor hygiene, a diet characterized by a low fiber intake and high levels of fat and sugar, a sedentary lifestyle, pollution, and various toxins can disrupt the natural balance of gut and salivary microbiota. Changes in microbiota composition, known as dysbiosis, can contribute to dysregulation of the immune system and the development of inflammatory diseases. To date, alterations in salivary and gut composition have been described in various diseases, including autoimmune, metabolic, allergic, psychiatric diseases, inflammatory bowel diseases, and malignant tumors. This study aimed to compare the composition and diversity of salivary and gut microbiota between CSU patients and healthy controls. Methodology: This case-control study included 22 CSU patients and 23 healthy controls. The diagnosis of CSU was established according to international guidelines for urticaria. Participants with certain conditions that could affect gut microbiota composition were excluded from further investigation. The exclusion criteria were as follows: participants who had taken systemic antibiotics and commercial probiotics in the last 3 months, patients with inflammatory bowel diseases, diabetes, obesity, psychiatric diseases or malignancy, participants who have been smoking more than 5 cigarettes per day, patients with periodontal diseases, oral ulcers, and oral inflammatory diseases and participants who were pregnant. All 45 participants were instructed to provide stool and salivary samples using commercial collection kits, according to the manufacturer’s instructions. Bacterial DNA was extracted from stool and saliva samples. 16S rRNA sequencing was performed to detect the salivary and gut microbiota composition of all subjects. Furthermore, blood samples from the patient group were taken to perform routine diagnostic tests (differential blood count, ESR, CRP, IgG anti-TPO, total IgE, vitamin D). CSU patients were also asked to fill out two questionnaires: Urticaria activity score (UAS) and Chronic urticaria quality of life (CU-Q2oL), to assess the activity of the disease and its impact on quality of life. Results: There were no significant differences in age or gender between the CSU group and healthy controls group. Half of the CSU patients had elevated anti-TPO antibodies, while 2/3 had vitamin D deficiency. There was a significant association between elevated anti-TPO and decreased total IgE (p<0.05), suggesting a type IIb autoimmune CSU. To evaluate alterations in the gut and salivary compositions and diversities between the two groups, we examined alpha diversity (measures the diversity of microbial species within a sample) and beta diversity (measures the differences in species composition between the samples). Alpha diversity of the gut microbiota was significantly reduced in CSU patients (p < 0.05) compared to healthy controls. Beta diversity revealed significant clustering (p < 0.05) between the CSU patients and healthy controls. Analysis of alpha and beta diversity of salivary microbiota didn´t reveal significant differences between the CSU group and healthy controls.To explore the statistically significant differences in the relative abundance of gut and salivary bacteria between the two groups, the linear discriminant analysis (LDA) effect size (LEfSe) was performed. LEfSe identified a statistically significant increased abundance of gut bacteria from the Lachnospiraceae family in the healthy group, including Lachnospira, Roseburia, Ruminococcus, Coprococcus, and Eubacterium eligens group (p<0.05). Members of the Lachnospiraceae family are among the major producers of short-chain fatty acids (SCFAs). These results indicated a notable decrease in the abundance of SCFAs-producing bacteria in the gastrointestinal tract of CSU patients. LEfSe analysis of salivary microbiota between CSU patients and healthy individuals showed statistically significant differences in the abundance of specific bacterial species, genera, families, and orders (p < 0.05). Bacteria from the order Burkholderiales, family Flavobacteriaceae, and genera Capnocytophaga, Kingella, and Lautropia were significantly more abundant in the CSU group compared to healthy controls. On the other hand, species Streptococcus sanguinis and Hemophilus pitmmaniae were significantly less abundant in CSU patients compared to healthy subjects. Furthermore, the receiver operating characteristic curve (ROC) was used to analyze the potential diagnostic value of statistically significant bacteria. Bacterial genera Lachnospiraceae NK4A136, Eubacterium eligens, and Roseburia in the gut, as well as genera Kingella and Lautropia and species Capnocytophaga leadbetteri in the saliva, were identified as potential diagnostic biomarkers for CSU. Analysis of the association between salivary and gut microbiota revealed that a higher number of bacteria originating from saliva were observed in CSU patients compared to healthy individuals. This indicates that in CSU patients, a higher quantity of these bacteria might be playing a role in immune system activation within the gut. This finding suggests that in CSU patients, an increased abundance of these bacteria may be contributing to immune system activation within the gut. Conclusion: Salivary and gut microbiota compositions significantly differ between CSU patients and healthy individuals. Significant changes in microbial compositions can contribute to the dysregulation of the immune system and result in an enhanced inflammatory response characteristic of CSU. These findings provide important insights into the potential role of microbiota in the development of CSU and highlight the need for continued research in this area.