This study investigated the effects of AgNPs on soil microbial communities when biosolids, containing AgNPs, were amended into soil. A field-collected sandy loam soil was amended with biosolids containing aged, PVP-coated AgNPs at measured concentrations of 3, 48, 123, 290 and 706 mg Ag kg−1 of soil (dry weight). The control treatment consisted of soil without AgNPs (Con-0) but amended with biosolids. An additional treatment of Ag+ (AgNO3) at 138 mg Ag kg−1 was included as appropriate. The AgNPs were aged in biosolids for 72 h before being spiked into soil. The impact of aged-AgNPs on the in-situ soil microbial community was assessed using a suite of microbial tests that assessed the effects on microbial growth, biomass, activity and diversity. The extractable Ag+ in AgNPs spiked soils increased with increasing AgNP concentrations and ranged from <0.1 to 3.38 mg Ag+ kg−1 soil. The extractable Ag+ in the AgNO3 treatment (i.e., 138 mg Ag kg−1) was 5 times lower than the comparable AgNP treatment (i.e., 123 mg Ag kg−1). A significant impact of AgNPs on the soil microbial community was observed with median inhibitory concentrations (IC50) ranging from 11 to >706 mg Ag kg−1. AgNP treatments showed a 10–64% reduction in microbial activities than the AgNO3 treatment at a similar total Ag concentration. The toxicity of AgNPs were more pronounced overtime, suggesting that Ag+ continued to be released from biosolid-aged AgNPs in the soil environment and likely contributed to the observed toxicity. Emergence of a silver tolerant bacterium (Rhodanobacter sp.) was observed in soils with low to moderately high AgNP concentrations (48 to 290 mg Ag kg−1). Our results suggest that AgNP exposure in soil amended with biosolids may have significant inhibitory effects on the soil microbial community despite their potential sulfidation in biosolid-amended soils.