Abstract: In order to provide excellent nitrogen-fixing bacteria strains for the subsequent development of biofertilizer, the leaves of pepper were taken as the research object and the selective incubation culture medium was used to separate and screen phyllosphere nitrogen-fixing bacteria, and determine their ability to N fixation, antagonistic ability against plant pathogenic. The species of strains were identified based on morphological characteristics and the phylogenetic analysis of partial 16S rDNA sequence. The results showed that two nitrogen-fixing strains of LJ-1（Acinetobacter sp.） and LJ-2（Rhizobium sp.） were isolated from the leaves of pepper and the nitrogenase activity was 93.26 and 102.23 U·L^-1, respectively. The two strains had broad-spectrum antifungal activity and acid-alkali resistance, and the inhibitory effects against Sclerotinia sclerotiorum, Botrytis cinerea, Rhizoctonia solani were more than 50%. The phyllosphere nitrogen-fixing strains LJ-1 and LJ-2 had nitrogen fixation and biocontrol functions, which could provide microbial resources for development of phyllosphere nitrogen-fixing bacteria fertilizer.