Background and aims: Bacillus spp. are sources of antifungal metabolites with the potential use of preventing fungal infection. This study was aimed to identify and isolate inhibitory bacterium against pathogenic fungi in Isfahan using molecular method.

Methods: In this cross-section descriptive, 150 samples (soil, air, and surface) from different parts of Isfahan were prepared and investigated the effect of inhibitory bacteria grown on nutrient agar medium on the growth of Aspergillus niger, Aspergillus flavus and Mucor hiemali. Qualitative investigation of fungal growth  inhibition was done by inoculation methods, and quantitative evaluation was conducted using linear culturing in distance of 0.5, 1, 1.5, 2, 2.5 and 3 cm of fungal suspension (10⁴ spore/µl) from the center of plates (bacterial inoculation,
0.5 Macfarland). Samples were incubated in 30 ^ͦC for 96 hours and identification of the inhibitory bacterium was done according to biochemical tests and molecular method of colony-PCR.

Results: The effect of inhibitory bacteria against Aspergillus niger, Aspergillus flavus and Mucor hiemali was from 0.5 to 3 cm. According to biochemical tests and colony-PCR, the bacterium with the most inhibitory effect was Bacillus atrophaeus strain HNSQJYH170.

Conclusion: Bacillus atrophaeus strain HNSQJYH170 is endemic in Isfahan and can be used in antibiotic production and biological control.