The antibacterial activities of isolates obtain from tempeh made from beans Igołomska variety fermented by Rhizopus oligosporus NRRL 2710 was assessed in this study. The antibacterial factor was estimated during 48 h fermentation. Bacillus subtilis DSM 347 was used as a test microorganism. The assessment of bacteria growth inhibition was performed by using several method: paper disk, agar slab well, nephelometric and measurement of impedance changes. Isolates obtain after 40h fermentation showed the highest inhibition of growth of test bacteria. The kind of hydrothermal treatment of bean seeds had an influence on the antibacterial activity.