The genus Peribacillus belongs to the family Bacillaceae and has recently been taxonomically separated from the genus Bacillus. Since species from the genus Bacillus play a significant role in maintaining terrestrial ecosystems and have great potential for agricultural applications, it is assumed that members of the Peribacillus genus also possess similar characteristics. The aim of this research is to investigate the potential of Peribacillus species isolated from a karst cave to inhibit the growth of human and plant pathogens and explore the role of these species in the spread of antibiotic resistance in nature. Sediment and water samples were collected from a cave near the settlement of Konjevrate in the Šibenik-Knin County and used for the isolation of bacteria. Gram-positive isolates that grew on R2 agar were identified using MALDI-TOF method. After identification, their resistance to five classes of antibiotics (carbapenems, glycopeptides, fluoroquinolones, macrolides/lincosamides and oxazolidinones) was tested by disk diffusion method. Antimicrobial activity was assessed against eight potentially pathogenic bacteria (E. coli, B. cereus, S. aureus, L. innocua, A. tumefaciens, E. amylovora, P. syringae and S. enterica) that cause diseases in humans and plants and on the phytopathogenic mold Botrytis cinerea. Out of 75 isolated Gram positive isolates, 11.3% were identified as Peribacillus spp. Out of 17 tested Peribacillus isolates, 35.3% were resistant to macrolides/lincosamides and 5.9% were resistant to fluoroquinolones and oxazolidinones, while none of the isolates showed resistance to carbapenems and glycopeptides. Peribacillus isolates showed no inhibitory activity against pathogenic bacteria or it was very weak in 5.9% of isolates. 47.1% of the isolates showed strong inhibitory activity against B. cinerea.