ABSTRACT Tomato is one of the most important vegetables in the world. Decay after harvest is a major issue in the development of tomato industry. Currently, the most effective method for controlling decay after harvest is storage of tomato at low temperature combined with usage of chemical bactericide; however, long-term usage of chemical bactericide not only causes pathogen resistance but also is harmful for human health and environment. Biocontrol method for the management of disease after tomato harvest has great practical significance. In this study, antagonistic bacterium B-6-1 strain was isolated from the surface of tomato and identified as Enterobacter cowanii based on morphological characteristics and physiological and biochemical features combined with sequence analysis of 16SrDNA and ropB gene and construction of dendrogram. Effects of different concentrations of antagonistic bacterium E. cowanii suspension on antifungal activity after tomato harvest were analyzed by mycelium growth rate method. Results revealed that antifungal activity was also enhanced with increasing concentrations of antagonistic bacterium; inhibitory rates of 1 × 105 colony-forming units (cfu)/mL antagonistic bacterial solution on Fusarium verticillioides, Alternaria tenuissima, and Botrytis cinerea were 46.31%, 67.48%, and 75.67%, respectively. By using in vivo inoculation method, it was further confirmed that antagonistic bacterium could effectively inhibit the occurrence of B. cinerae after tomato harvest, biocontrol effect of 1 × 109 cfu/mL zymotic fluid reached up to 95.24%, and antagonistic bacterium E. cowanii has biocontrol potential against B. cinerea after harvest of fruits and vegetables.