We conducted an experiment with two agricultural soils with acidic and alkaline pH levels to assess the effects of zinc oxide nanoparticles (nZnO) on the bacterial community. The effect of the nZnO concentrations (0, 0.1, 1, 10, 100, 1000 mg Zn/kg soil) and contact time between nanoparticles and soil (180 days) was considered. We measured the microbial respiration rate, nitrogen transformation, enzymatic activities (dehydrogenase (DH), acidic phosphatase (ACP), and alkaline phosphatase (ALP)), and the community-level physiological profile (CLPP) soil parameters. Respiration potential and nitrogen transformation were negatively affected only at the highest nZnO concentration. The changes in enzymatic activities were very variable with time and between both soils. A stimulating effect of the nanoparticles on microbial activity was clearly shown at 30 days after the nZnO application in both soils, except for the 1000 mg/kg in calcareous soil, after which time in the latter, the functional richness of the bacterial community was reduced to virtually zero. However, values of the enzymatic activities demonstrated that there was self-adaptation of microbial communities over the study period (180 days). The nZnO 1000 mg/kg dose produced an increase in bacterial growth in the acidic soil, without apparent changes in their metabolic profiles over time. A good correlation was found between microbial respiration rates (calcareous and acidic soils) and microbial metabolic activity (acidic soil) based on extracted Zn concentrations. Our findings suggest the necessity of additional studies to examine the effects of nZnO in natural microorganism populations in soil with different pH levels for extended periods of time.