Roadside soil communities include a diverse array of microbes and invertebrates that play vital roles in nutrient cycling, soil and vegetation health, and food webs for terrestrial wildlife. These communities are also susceptible to exposure to road-related chemical stressors, such as dust suppressants and soil stabilizers. Although dust suppressants are commonly applied to unpaved roads worldwide, the effect of these chemicals on soil health and the potential toxicity to roadside organisms has been understudied. To address this gap, we exposed soil microbial communities present in a natural silt loam to two application rates of two dust suppressant products: durablend™, a magnesium chloride plus binder, and EnviroKleen®, a synthetic fluid plus binder. One application rate simulated direct overspray and the other was half the direct overspray rate. We used the same two products and rates in experimental treatment of sand substrates in 7-day toxicity tests with terrestrial isopods (Porcellio scaber). In natural soils, phospholipid fatty acid analysis (PLFA) indicated shifts in microbial communities with product application that persisted throughout the 28-day experiment. Treatment with durablend™ decreased the abundance of fungi, while treatment with EnviroKleen® increased the abundance of gram-positive bacteria, relative to untreated controls. Treatment with either product decreased the abundance of actinomycetes. In isopod tests, survival was reduced relative to the untreated sand at both durablend™ rates and at the direct overspray rate of EnviroKleen®. These results provide the first experimental evidence that exposure to dust suppressant chemicals can alter the composition of soil microbial communities and cause mortality of terrestrial invertebrates. The most pronounced effects occurred at the direct overspray rate, underscoring the need for responsible application procedures in the field. Overall, these results help address a general lack of information on environmental impacts of dust suppressant and soil stabilizer applications and can be used to help inform management decisions for the more than 14 million km of unpaved roads worldwide.