In this study, we investigated the effects of one-off applications of glyphosate, glufosinate, paraquat, and paraquat-diquat on soil microbial diversity and function. All herbicides were added to soil as pure compounds at recommended dose and were incubated under laboratory conditions for 60 days. High-throughput phylogenetic marker gene sequencing revealed that none of the herbicides significantly influenced the richness, evenness and composition of bacterial and archaeal communities. Likewise, the diversity, composition and size of nematode communities were not significantly influenced by any of the herbicides. From a functional perspective, herbicides did not significantly affect fluorescein diacetate hydrolysis (FDA) and beta-glucosidase activities. Furthermore, the ability of soil organisms to utilise 15 substrates was generally unaffected by herbicide application. The only exception to this was a temporary impairment in the ability of soil organisms to utilise three organic acids and an amino acid. Given the global and frequent use of these herbicides, it is important that future studies evaluate their potential impacts on microbial communities in a wider-range of soils and environmental conditions.