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Organophosphorus compounds have an extensive history as both agricultural pesticides as well as chemical nerve agents. Decades of research have demonstrated numerous links between these chemicals and their direct and indirect effects on humans and other organisms. The inhibitory effects of organophosphate pesticides (OPPs) on metazoan physiology, are well-characterized; however, the effects of organophosphorus compounds on soil microbes - essential contributors to key agricultural processes - are poorly understood. Chlorpyrifos (CPF) is an OPP that is used globally for crop protection. Studies of CPF application to soils have shown transient effects on soil microbial communities with conflicting data. Here, we directly test the effect of CPF on a panel of 196 actinobacteria strains, examining the effects of CPF on their growth and in vitro phenotypes on solid media. Strains were grown and replica-plated onto media containing CPF or a vehicle control and grown at 28°C. CPF dramatically inhibited the growth of most strains and/or altered colony morphologies, with 13 strains completely inhibited by CPF. In disk diffusion assays with CPF, its degradation product 3,5,6-trichloropyridinol (TCP), malathion, parathion, monocrotophos and mevinphos, only CPF exhibited direct antimicrobial activity suggesting that the observed effects were due to CPF itself.IMPORTANCEChlorpyrifos is a globally used pesticide with documented neurological effects on non-target organisms in the environment. Finding that chlorpyrifos can inhibit the growth of some soil microbes in vitro may have implications for the composition, stability, and health of the soil microbiome. Due to the importance of soil microbes to numerous biogeochemical processes in agricultural systems, additional investigations into the non-target effects of CPF on soil microbes are clearly needed.