Soil is a classical source of beneficial microorganisms. Soil microbiomes provided the overwhelming majority of antibiotic-producing strains, biocontrol agents, probiotics, and plant-protecting bacteria. The functionality of strains isolated from various soil samples is predetermined by the biosynthetic potential encoded in their genomes. Here, we describe a novel Peribacillus simplex d27.3 strain isolated from the soil sample of a pine forest in the Republic of Dagestan, Russia. P. simplex d27.3 displayed antibiotic activity against gram-positive bacteria and fungi while being inactive against the model hypersensitive gram-negative strain E. coli ΔlptD. Metabolomic analysis revealed that antimicrobial activity was partially mediated by the fengycin lipopetides (C-16 fengycin A, C-17 fengycin A, and C-16 fengycin B). In addition, the P. simplex d27.3 strain was found to produce other hydrophilic and more hydrophobic antimicrobials yet to be described. Thus, the P. simplex d27.3 strain is a producer of useful antimicrobial compounds with a high potential for application in biotechnology and agriculture. Copyright: © 2024 Baranova et al.