Mesotrione is a widely used agricultural herbicide and is frequently used alone or as an adjuvant for the herbicides glyphosate and atrazine. The effects of mesotrione are largely untested on beneficial non-target species such as spiders. Different spider species may be differentially susceptible due to size differences, microhabitat, and levels of exposure to this herbicide via soil contact. We tested mortality differences of seven species of spider when exposed to field-relevant concentrations of mesotrione-treated soil over a 55-day period. We tested the web-building spiders Frontinella pyramitela and Tetragnatha laboriosa. We also tested the stem and leaf-dwelling ambush spiders Mecaphesa asperata and Pisaurina mira and three species of ground-dwelling wolf spiders that vary in their burrowing propensities: Hogna lenta (infrequent burrower), Tigrosa helluo (facultative burrower) and a habitually burrowing wolf spider Trochosa ruricola. All seven of these species commonly occur in mesotrione-treated agricultural systems. We found that the web-building spider Frontinella but not Tetragnatha showed increased mortality compared to control treatments. Mecaphesa, Pisaurina, Trochosa, and Tigrosa all showed large significant increases in mortality under chronic exposure to mesotrione-treated soil whereas the wolf spider Hogna lenta was unaffected. We also found sex-specific mortality effects in Pisaurina with males having higher mortality. Several species showed significant shifts in space use when exposed to mesotrione and we found significant interaction between spider weight gain and herbicide treatment. In general, mesotrione is an unsafe herbicide for some species of spiders. Alternative herbicides to mesotrione should be considered to minimize the negative biocontrol impact on beneficial spiders within integrated pest management systems