Improving the ecological functions of barren soils by organic manure is an important technology in soil fertility management. The effect of organic manure or biochar with equal carbon input was compared on soil microbial diversity and activity by using a field experiment in a red soil derived from Quaternary red clay in Subtropical China. Soil microbial diversity was measured by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE), than soil CO2 flux was observed to analyze its relationship with soil microbial diversity. The two-year experimental results showed that manure application altered significantly soil microbial community composition by changing soil properties. Applying organic manure and biochar increased the bacterial diversity, but decreased the fungal diversity in the second year. Fertilization could raise soil CO2 flux with the highest value under mixed application of organic manure and straw. Correlation analysis showed that soil respiration rate was significantly positively correlated to bacterial and fungal diversity, in which soil bacterial diversity played more contributions (71%) than fungal diversity (29%). Aggregated boosted tree (ABT) analysis showed that the relative contribution of soil phosphorus contents (total and available phosphorus content) to the variation of bacterial and fungal diversity were 44.8% and 47.4%, respectively, suggesting that different fertilization treatments influenced soil phosphorus supply and then drove the changes of microbial diversity. In total, mixed application of organic manure and straw can quickly improve red soil microbial diversity and functions.