Abstract Background The increasing production of nanoplastics and the fragmentation of microplastics into smaller particles suggest a plausible yet unclear hazard in the natural environment, such as soil. We investigated the short-term effects (28 days) of polystyrene nanoparticles (PS-NPs) on the activity and biomass of soil microbiota, and the functional diversity of soil enzymes at environmental relevant low levels in an incubation experiment. Results Our results showed a significant decrease in microbial biomass in treatments of 100 and 1000 ng PS-NP g−1 DM throughout the incubation period. Dehydrogenase activity and activities of enzymes involved in N-(leucine-aminopeptidase), P-(alkaline-phosphatase), and C-(β-glucosidase and cellobiohydrolase) cycles in the soil were significantly reduced at day 28 suggesting a broad and detrimental impact of PS-NPs on soil microbiota and enzymes. Leucine-aminopeptidase and alkaline-phosphatase activities tended to decrease consistently, while β-glucosidase and cellobiohydrolase activities increased at high concentrations (e.g., PS-NP-1000) in the beginning of the incubation period, e.g., at day 1. On the other hand, basal respiration and metabolic quotient increased with increasing PS-NP application rate throughout the incubation period possibly due to increased cell death that caused substrate-induced respiration (cryptic growth). Conclusions We herewith demonstrated for the first time the potential antimicrobial activity of PS-NPs in soil, and this may serve as an important resource in environmental risk assessment of PS-NPs in the soil environment.