[Objective] By analyzing the effects of Bacillus subtilis antibiotics, spores and fermentation on the microorganism quantity and microbial community in cucumber phyllosphere, providing theoretical basis for the effective application. [Methods] With cucumber as the experimental material, setting up eight treatments: applying the antibiotics, spores and fermentation before or after inoculating Botrytis cintrea, only inoculating Botrytis cintrea and spraying the sterile water. The experiment was conducted in the indoor. Using the method of traditional microbial separation to count the number, the cultured bacteria, actinomycetes and fungi of cucumber phyllosphere were respectively separated with PB, High’s No.1 and PDA medium. At the same time, analyzing the phyllosphere microorganism samples of different treatments by the Dcode Universal Mutation Detection System for DGGE, the quantitative analysis of strip number and gray level were carried on by Quantity One analysis software, the various belt was clustered by UPGMA method, the microorganism quantity and microbial community was analyzed by microbe richness index, Shannon-Wiener diversity index and Pielou index, the differential DNA sequencing results were analyzed by NCBI Blast program homology, and clustered by The Sequence match program. [Results] The results of different treatments on the microorganism quantity and microbial community in cucumber phyllosphere, showed that spraying before inoculation with the Botrytis cintrea is the best applying way of Bacillus subtilis antibiotics, spores and fermentation, can effectively reduce the number of culturable fungi, increase the number of cultured bacteria and actinomycetes, increase leaf microbe richness index, Shannon-Wiener diversity index and Pielou index. Antibiotic treatment on the occasion of leaf microbial population structure is minimized. [Conclusion] Bacillus subtilis BSD-2 fermentation can reduce the number of culturable fungi. Bacillus subtilis BSD-2 spores can increase the number of the cultured bacteria and actinomycetes. Bacillus subtilis BSD-2 antibiotics can keep phyllosphere microbial richness and diversity index.