Reclaimed water (RW) is an alternative water resource that has been utilized all over the world, but its environmental effects are not fully understood. Soil biodiversity is an important indicator of soil tolerance and resilience. In the present study, the impact of RW irrigation on the microbial community diversity and chemical properties of topsoil was investigated by monitoring nitrogen (N) rates. Tomato plants were grown on plots which had been irrigated with reclaimed water for 5 years with varying levels of N fertilization (N270, 270 kg ha−1; N216, 216 kg ha−1; N189, 189 kg ha−1; and N135, 135 kg ha−1). Soil bacterial community composition was analyzed by PCR amplification of the 16S rDNA gene and Illumina MiSeq high-throughput sequencing technology of a total of 770,066 quality sequences. The results showed that long-term RW irrigation altered the bacterial composition of soil in an N-dependent manner. RW irrigation increased the abundances of Gemmatimonadetes, Actinobacteria, Firmicutes, and Nitrospirae in soils. The Chao, ACE, and H indices revealed no significant difference under RW irrigation with varying levels of N fertilization. The tomato yield and partial factor productivity from applied N for RN216 increased significantly under RW irrigation with reducing N fertilization. RW irrigation increased the yield of tomato and the abundance of functional microorganisms, which eventually improved the practice of irrigating with reclaimed municipal wastewater. Meanwhile, the potential environmental and health risks of long-term RW irrigation warrant further investigation.