Bacteria play vital roles in soil biological fertility; however, it remains poorly understood about their response to long-term fertilization in Chinese Mollisols, especially when organic manure is substituted for inorganic nitrogen (N) fertilizer. To broaden our knowledge, high-throughput pyrosequencing and quantitative PCR were used to explore the impacts of inorganic fertilizer and manure on bacterial community composition in a 35-year field experiment of Chinese Mollisols. Soils were collected from four treatments: no fertilizer (CK), inorganic phosphorus (P) and potassium (K) fertilizer (PK), inorganic P, K, and N fertilizer (NPK), and inorganic P and K fertilizer plus manure (MPK). All fertilization differently changed soil properties. Compared with CK, the PK and NPK treatments acidified soil by significantly decreasing soil pH from 6.48 to 5.53 and 6.16, respectively, while MPK application showed no significant differences of soil pH, indicating alleviation of soil acidification. Moreover, all fertilization significantly increased soil organic matter (OM) and soybean yields, with the highest observed under MPK regime. In addition, the community composition at each taxonomic level varied considerably among the fertilization strategies. Bacterial taxa, associated with plant growth promotion, OM accumulation, disease suppression, and increased soil enzyme activity, were overrepresented in the MPK regime, while they were present at low abundant levels under NPK treatment, i.e. phyla Proteobacteria and Bacteroidetes, class Alphaproteobacteria, and genera Variovorax, Chthoniobacter, Massilia, Lysobacter, Catelliglobosispora and Steroidobacter. The application of MPK shifted soil bacterial community composition towards a better status, and such shifts were primarily derived from changes in soil pH and OM.