Four species from three families of spiders were exposed to sublethal concentrations of the neurotoxic pesticide malathion: Schizocosa ocreata (Hentz 1844), Rabidosa rabida (Walckenaer 1837), Frontinella communis (Hentz 1850), and Salticus scenicus (Clerck 1757). Spider activity was recorded using a proprietary computer vision system equipped with artificial intelligence routines. Exposure to malathion changed the spiders' propensity to move, levels and patterns of activity, and distance moved. Dosed spiders increased their activity between 12 and 40%, depending on the species. Continuous recordings for ≥ 24 h revealed the peak activity for dosed R. rabida and S. scenicus was shifted ∼ 1 h earlier than controls. Spiders exposed to malathion also significantly increased the distance they moved per locomotory bout. This is consistent with the action of an organophosphate neurotoxin acting as an acetylcholinesterase inhibitor. Thus, exposure to sublethal doses of malathion appears to affect the neural basis for these spider's normal diel periodicities, time budgets, and patterns of locomotion, probably reducing their efficiency as agents of biological control.