Beauveria bassiana is a well-known entomopathogenic fungi with worldwide distribution that can be used as a microbial pesticide. To date, studies of B. bassiana have focused on its insecticidal mechanism, symbiosis with plants and antagonism of plant pathogens; however, few studies of its influence on the microecological phyllosphere and at physiological level of the plant have been conducted. To investigate the ecological security of B. bassiana in the paddy phyllosphere, we evaluated the effects of different concentrations of B. bassiana spore suspensions and a chemical pesticide (acephate) on paddy phyllosphere microbial flora and protective enzyme activities by a potted trial. B. bassiana were then induced to express the egfp gene (green fluorescent protein gene) through transformation with a vector, after which real-time polymerase chain reaction (PCR) was used to quantify the organisms and measure their dynamics in a paddy phyllosphere. We also evaluated the effects of different concentrations of B. bassiana suspensions on the phyllosphere microflora using DGGE. To accomplish this, B. bassiana specific DNA primers were designed based on the green fluorescent protein sequence marked B. bassiana. Amplification of B. bassiana DNA using the eGPF-F1/eGFP-R1 primers yielded a single 289bp-long product with a detection limit of 10fg/μL of B. bassiana genomic DNA. The pot experiments, which were conducted in the botanical garden of Nanjing Normal University, consisted of the following seven treatments: sterile water applied as a control (CK), inoculation with the larva of Chilo suppressalis (A), application of the B. bassiana spore suspension at 7.5×10⁴spores/ml (B), 7.5×10⁵ spores/ml (C), 7.5×10⁶ spores/ml (D), or 7.5×10⁷ spores/ml (E), application of acephate emulsifiable concentrates (F). Each treatment group was covered with gauze (3 m × 0.7 m × 1.5 m) after treatment. Fluorescence quantitative PCR analysis of the rice phyllosphere DNA revealed that B. bassiana was maintained for at least 30 days on the leaves of paddy plants after application, but when a higher initial dosage of the B. bassiana was applied, the B. bassiana population on the phyllosphere decreased more rapidly. When compared with chemical pesticide, B. bassiana did not significantly affect the antioxidant activity of three enzymes in paddy leaves. Specifically, the enzymatic activities of SOD and POD in B. bassiana treatment groups were 20.38 and 8.65% higher, respectively, than those in the CK group on the tenth day, while the activity of CAT was 33.67% higher than that of the CK group on day 30. However, the enzymatic activity of CAT in the F group was 42.71% lower than that of the CK group on day 10. DGGE cluster analysis showed that B. bassiana did not significantly influence the bacterial or fungal community structures on the paddy phyllosphere, and the microbial community structure similarity, Shannon index and band number in the B. bassiana treated group were higher than in the negative control group. These results indicated that B. bassiana is an environmental friendly microbial pesticide. Future studies of B. bassiana should focus on its effects on other endophytes in plants. Studies to evaluate the role of B. bassiana could lead to a new paradigm on its successful use in biological control programs.