Urban development is one of the leading causes of biodiversity change. Understanding how soil microorganisms respond to urbanization is particularly important because they are crucial for the provisioning of ecosystem functions and services. Here, we collected monthly soil samples over one year across three locations representing an urbanization gradient (low-moderate-high) in the arid Southwestern USA, and we characterized their microbial communities using marker gene sequencing. Our results showed that microbial richness and community composition exhibited nonsignificant changes over time regardless of the location. Soil fungal richness was lower in moderately and highly urbanized locations, but soil bacterial/archaeal richness was not significantly different among locations. Both bacteria/archaea and fungi exhibited significant differences in community composition across locations. After inferring potential functional groups, soils in the highly urbanized location had lower proportions of arbuscular mycorrhizal fungi and soil saprotrophic fungi but had higher proportions of bacterial taxa involved in aromatic compound degradation, human pathogens, and intracellular parasites. Furthermore, ammonia-oxidizing bacteria were more abundant in the highly urbanized location, but ammonia-oxidizing archaea were more abundant in lowly and moderately urbanized locations. Together, these results highlight the significant changes in belowground microbial communities across an urbanization gradient, and these changes might have important implications for aboveground–belowground interactions, nutrient cycling, and human health.