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Soil microbial communities play an important role in nutrient cycling; however, their response under repeated long-term fertilization has attracted little attention and needs further appraisal. A 14-day incubation study compared the relative abundance, diversity, and composition of bacterial and fungal microbial communities in soils treated with long-term applications of chemical fertilizer (CF), pig manure plus chemical fertilizer (PMCF), and rice straw plus chemical fertilizer (SRCF) in a paddy field. A high-throughput sequencing approach was applied to assess the diversity and composition of microbial community. Results revealed the Shannon index of the bacterial community decreased with fertilizer addition but increased in case of fungal community. The abundance of the <i>Actinobacteria</i> was higher in the PMCF, while <i>Proteobacteria</i> were higher in the CF and SRCF treatments than those in the unamended control under alternate wetting and drying (AWD) and permanent flooding (PF). In addition, chemical fertilizer history increased the abundance of <i>Firmicutes</i> under AWD. Initially, <i>Nitrospira</i> were found higher in the unamended control than in the amended treatments, but an increase was observed with time in fertilized treatments. Among all genera, <i>Proteobacteria</i> were the most abundant bacterial genus. The main properties that markedly affected the bacterial communities were SOC (<i>R</i>² = 0.4037, <i>p</i> < 0.02), available P (<i>R</i>² = 0.3273, <i>p</i> < 0.05), and NO₃⁻ (<i>R</i>² = 0.3096, <i>p</i> < 0.08). Soil physicochemical factors and biogenic factors explained a variation of 46.27% and 29.35%, respectively. At the same time, 4.59% was the combined effect of physicochemical and biogenic factors. Our results suggested that the physicochemical properties had a more significant impact on bacterial activities than water regime by increasing N and organic matter concentrations in the soils.