To assess the impact of silver nanoparticles (SNP) on soil microbial biomass, microbial activity, and enzyme activities, a medium-term experiment over four months was performed in which soil was applied with increasing SNP-application rates compared to a control. The treatments included a single SNP-application dose analogous to 3.2 (SNP-1), 32 (SNP-10), and 320 (SNP-100) μg Ag (kg dry soil)–1 and a control without SNP application, respectively. At the end of the experiment, clear evidence was found that microbial biomass was significantly decreased with increasing SNP-application rate, while basal respiration was increased in this direction. In addition, metabolic quotients were increased in the SNP treatments compared to the control. This is at least circumstantial evidence that the efficiency of substrate use was lowered in SNP-treated soils. Another suggestion might be that after four months microbial-community composition was changed due to SNP. No treatment effects were found for microbial biomass N, fluorimetric enzymes, and the abiotic soil parameters pH and soil organic C.