Soil microorganisms are key contributors to nutrient cycling and are essential for the maintenance of healthy soils and sustainable agriculture. Although the antimicrobial effects of a broad range of nanoparticulate substances have been characterised in vitro, little is known about the impact of these compounds on microbial communities in environments such as soil. In this study, the effect of three widely used nanoparticulates (silver, silicon dioxide and aluminium oxide) on bacterial and fungal communities in an agricultural pastureland soil was examined in a microcosm-based experiment using a combination of enzyme analysis, molecular fingerprinting and amplicon sequencing. A relatively low concentration of silver nanoparticles (AgNPs) significantly reduced total soil dehydrogenase and urease activity, while Al2O3 and SiO2 nanoparticles had no effect. Amplicon sequencing revealed substantial shifts in bacterial community composition in soils amended with AgNPs, with significant decreases in the relative abundance of Acidobacteria and Verrucomicrobia and an increase in Proteobacteria. In particular, the relative abundance of the Proteobacterial genus Dyella significantly increased in AgNP amended soil. The effects of Al2O3 and SiO2 NPs on bacterial community composition were less pronounced. AgNPs significantly reduced bacterial and archaeal amoA gene abundance in soil, with the archaea more susceptible than bacteria. AgNPs also significantly impacted soil fungal community structure, while Al2O3 and SiO2 NPs had no effect. Several fungal ribotypes increased in soil amended with AgNPs, compared to control soil. This study highlights the need to consider the effects of individual nanoparticles on soil microbial communities when assessing their environmental impact.