A study on the effects of minimal inhibitory concentration of several antibiotics, including Gentamicin, Streptomycin, Ribostamycin Sulfate, and Sagamicin Sulfate, on pollution microbes of L-malic acid was conducted in this paper. The experiment of the simulation ferment process with the same temperature, pH value and ventilation conditions in vibration culture with twice pollution microbes was carried out added and the results were validated by agar plate culture. It was showed that Gentamicin had the strongest inhibitory effectiveness and Sagamicin Sulfate the second. The minimal inhibitory concentration of Gentamicin was 0.4 mg/L, and after twice pollution microbes added, the effective concentration was 0.8 mg/L. The result was used for the ferment processing practically with the fermentation tanks of 240 and 1700 L, and proved to be valid for L-malic acid fermentation.