During the screening of antiviral substances having inhibitory effects on tobacco mosaic virus (TMV) infection to tobacco plants, we found a bacterial isolate KTB3, which was identified as Acinetobacter sp., strongly inhibited the formation of TMV local lesions. When the culture filtrate from KTB3 was applied on the upper surface of leaves of Xanthi-nc tobacco plants at the same time of or 24 hours before TMV inoculation, almost complete inhibition was achieved. And 86% inhibition was shown when the culture filtrate was applied to the under surface of leaves. In field trials, transmission of TMV from diseased seedlings to healthy ones during transplanting work was reduced by 92%, when the culture filtrate was sprayed on to the tobacco seedlings, cv. NC82, 24 hours before transplanting. No toxic effects was observed on the tobacco plants. Antiviral substances from the culture filtrate was purified by ethanol precipitation, dialysis, DEAE-cellulose, and Sephadex G75 gel column chromatography. The purified active materials, which showed positive color reaction to sugar and protein inhibited TMV infection by 60% at 1µg/ml.