Gray mold of strawberries (Botrytis cinerea) represents a significant threat to strawberries, leading to a serious reduction in yield, especially in conditions of high humidity and abundant rainfall. The potential antimicrobial and antifungal properties of cannabinoids, and the development of new cannabidiol (CBD) nanoparticle formulations, could enhance strawberry protection during their cultivation and storage. The aim of this research is to understand how biopolymer nanoparticles loaded with CBD affect the growth and development of the phytopathogenic fungus Botrytis cinerea. Given the increasing problem of fungal pathogen resistance to traditional antifungal agents, scientists are searching for new alternatives and strategies. One of the new methods used in the suppression of phytopathogenic fungi is the encapsulation of compounds in biopolymer nanoparticles, achieving their timed release and prolonged action. Cannabinoids and related compounds from the Cannabis sativa plant exhibit significant pharmacological and agronomic properties, with some of them being explored as potential antifungal agents in agriculture. The CBD isolate encapsulation process was carried out using the ionic gelation method at the Institute of Chemistry, and the antifungal activity of optimal formulations of biopolymer nanoparticles loaded with CBD was tested on the phytopathogenic fungus Botrytis cinerea at the Institute of Phytopathology. This research confirms the fungistatic and potentially fungicidal activity of CBD nanoparticles against Botrytis cinerea isolated from infected strawberry fruits. The control sample recorded a high percentage of mycelium growth surface at 86%. Treatment with 1% CBD biopolymer nanoparticles showed mycelium growth at 20%, while treatment with 2% CBD nanoparticles yielded an average value of 25%. The mean value of the 3% CBD nanoparticle treatment resulted in the lowest mycelium-covered surface at 15%. Further research is needed to conduct additional studies with different concentrations and in vivo experiments. Additionally, this research suggests the potential use of biopolymer CBD nanoparticles as an environmentally friendly antifungal agent in strawberry production and storage.