Biochar has the potential to enhance microbial-mediated phosphorus (P) cycling in soils, but the underlying mechanisms remain largely unknown. We hypothesized that biochar amendment could enhance the production of acid and alkaline phosphomonoesterase, phosphodiesterase and P mineralization, which may vary depending on the P input. To test this hypothesis, we assessed the impacts of rice straw biochar application (0 and 4%) under different P-input rates (0, 30 and 90 kg P ha−1) on the relationships among P fractions, phosphatase activities and alkaline phosphomonoesterase-encoding bacterial (phoD gene) communities in an acidic soil. Biochar application under low P input (< 30 kg P ha−1) significantly increased the activities of phosphodiesterase and alkaline phosphomonoesterase but not that of acid phosphomonoesterase and depleted organic P. The results from the structural equation model revealed a dominant role of alkaline phosphomonoesterase in P mineralization. The increase in alkaline phosphomonoesterase activity was not related to an increase in phoD gene abundance but was due to a shift in community composition, which was primarily driven by the soil C:P ratio. Microbial network analysis demonstrated a more complex phoD gene community with more functionally interrelated groups as a result of biochar application under low P input than under high P input. Moreover, the specific enrichment of Micromonosporaceae under C-rich and P-poor conditions may play a critical role in alkaline phosphomonoesterase production and potential P mineralization. In conclusion, we demonstrated that biochar application under low P input supports a more organized phoD gene community and preferentially enriches taxa in terms of their capacity for P mineralization, which in turn may enhance P bioavailability and plant P acquisition.