Moisture and temperature play important roles in the assembly and functioning of prokaryotic communities in soil. However, how moisture and temperature regulate the function of niche- versus neutral-based processes during the assembly of these communities has not been examined considering both the total microbial community and the sole active portion with potential for growth in native subtropical grassland. We set up a well-controlled microcosm-based experiment to investigate the individual and combined effects of moisture and temperature on soil prokaryotic communities by simulating subtropical seasons in grassland. The prokaryotic populations with potential for growth and the total prokaryotic community were assessed by 16S rRNA transcript and 16S rRNA gene analyses, respectively. Moisture was the major factor influencing community diversity and structure, with a considerable effect of this factor on the total community. The prokaryotic populations with potential for growth and the total communities were influenced by the same assembly rules, with the niche-based mechanism being more influential in communities under dry condition. Our results provide new information regarding moisture and temperature in microbial communities of soil and elucidate how coexisting prokaryotic populations, under different physiological statuses, are shaped in native subtropical grassland soil.