Background: Biological control of fruit postharvest diseases by antagonistic microorganisms has been considered an effective alternative to chemical fungicides. The influence of microbial antagonists on fruit-associated microbiome will provide a new perspective for in-depth study of the antagonistic mechanism. In this study, the biocontrol efficacy of A. pullulans S-2 against postharvest diseases of tomatoes was investigated. Meanwhile, the fungal and bacterial microbiota on tomato surfaces were examined by high-throughput sequencing. Results: A. pullulans S-2 can significantly inhibit the decay rate, maintain fruit firmness and reduce weight loss of tomatoes. In addition, the treatment group can maintain higher titratable acid, ascorbic acid and lycopene than the control group. After using A. pullulans S-2, more dramatic changes were observed in fungal diversity than bacterial in the microbiota. Aureobasidium was significantly enriched in the treatment group, while Cladosporium, Mycosphaerella, Alternaria and Penicillium were deficient compared with the control group. Pantoea, Brevibacterium, Brachybacterium, Serratia, Glutamicibacter and Pseudomonas also had significant differences between the two groups.Conclusions: This study demonstrated that the application of A. pullulans S-2 resulted in alterations in the bacterial and fungal community and that could inhibit pathogens and decrease fruit disease incidence. It provides new insights into the dynamics of the tomato's surface microbiome after microbial antagonist treatment.