Abstract Background Multi-drug resistant bacteria hazards to the health of humans could be an agent in the destruction of human generation. Natural products of Bacillus species are the main source to access progressive antibiotics that can be a good candidate for the discovery of novel antibiotics. Wild honey as a valuable food has been used in medicine with antimicrobial effects. Objective Bacillus strains isolated from wild honey were evaluated for the potential antimicrobial activity against human and plant bacterial and fungal pathogens. Methods Three bacterial isolates were identified as strain Khuz-1 (98.27% similarity with Bacillus safensis subsp. Safensis strain FO-36b T ), strain Khuz-2 (99.18% similarity with Bacillus rugosus strain SPB7 T ), and strain Khuz-3 (99.78% similarity with Bacillus velezensis strain CR-502 T ) by 16S rRNA gene sequences. The strains were characterized by their ability to inhibit the growth of human and phytopathogenic fungi. Results The results indicated that B. rugosus strain Khuz-2 inhibited the growth of phytopathogenic and human fungal more effective than other ones. It seems that the strain Khuz-2 has a suitable antimicrobial and antifungal potential as a good candidate for further pharmaceutical research. Conclusion Based on the results of GC–MS, Pyrrolo [1,2-a] pyrazine-1,4-dion, hexahydro-3-(2-methylpropyle) (PPDHM) was the major compound for all strains which have a various pharmacological effect. Isolation and identification of beneficial bacteria from natural sources can play an important role in future pharmaceutical and industrial applications.