Biological nitrogen fixation complements nitrogen from fertilizers in crop plants under natural conditions. It also contributes to the reduction in chemical fertilizer (CF) utilization in cultivated lands, which fits the concept of sustainable agriculture. From this viewpoint, however, it is still unknown in paddy fields how soil bacterial nitrogenase and microbiota are affected by applied materials in the soil. Therefore, in this study, the effects of long-term material applications on potential nitrogenase activity and the microbiota of soil bacteria were investigated. The nitrogenase activity tended to be higher in manure compost (MC)-applied soils than in CF-applied soils in both summer and winter. Soil bacterial alpha diversity increased whereas soil ammonia availability decreased with the MC application. The dynamic response of soil bacterial microbiota was caused by the MC application. The abundance of Nitrospira, a class of ammonia and nitrite oxidation bacteria, was lower and the abundance of alpha-Proteobacteria was higher in the MC-applied soils than in the CF-applied soils. These results suggest that the alpha diversity increase and restricted availability of NH3-N might contribute to the increase in potential nitrogenase activity in the long-term MC-applied soils.