The effects of Penicillium tomii PT95 strain on Staphylococcus aureus, Bacillus subtilisus, Escherichia coli, Saccharomyces cerevisiaeae, Rhizopus nigricansa, Lactobacillus delbrueckii, Lactobacillus plantarum were studied by filter paper method and oxford cup method, the methods can screen out the most effective strain. At the same time, the minimum concentration of fermentation filtrate of Penicillium thomii was determined. Taking the diameter of bacteriostatic ring as an index, single factor test was used to optimize the culture conditions of carbon source, nitrogen source and inorganic salt etc. The results showed that Penicillium thomii PT95 strain had the most inhibitory effect on Bacillus subtilis, with the largest inhibition rate and up to 65.4%. The optimized medium was glucose 8.64?g/L, peptone 5.00?g/L, MgSO4·7H2O 0.30?g/L, NaCl 3.00?g/L, agar 20.00 g/L, pH7.0～7.2. Inhibitory substance was found abundantly in secondary metabolites of Penicillium thomii PT95 strain. Antibacterial activity was improved by optimal production conditions.