The objective of this study was to access the effect of long-term fertilization and film mulching application on soil properties and bacterial community structure. We used 16S rRNA gene to investigate soil bacterial community composition by high through-put sequencing. The results demonstrated that predominant groups in the bacterial community were: Proteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria, Firmicutes, Planctomycetes, Gemmatimonadetes, Verrucomicrobia, Chloroflexi and Cyanobacteria. Long-term fertilization of combined manure and nitrogen application caused significant decrease in soil bacterial diversity and richness compared to non-fertilization control, though manure fertilization alone played a significant role in restoring bacterial diversity. Film mulching and manure fertilization significantly increased the relative abundances of soil bacterial groups mentioned above. Furthermore, film mulching played significant role in shaping the bacterial community structure regardless of fertilization. Redundancy analysis (RDA) showed that soil moisture content, pH, total N and soil organic C had significant (P < 0.05) influence on dominant bacterial groups. Altogether, plastic film mulching and manure application prevented loss of soil bacterial diversity and abundance during long-term fertilization. These findings showed the detrimental use of combined manure and nitrogen fertilization to soil microbes and the useful application of manure fertilization coupled with film mulching to soil biodiversity in long-term fertilization experiments.