Organophosphorus (OP) and thiocarbamate (TC) agrochemicals are used worldwide as insecticides, herbicides, and fungicides, but their safety assessment in terms of potential off-targets remains incomplete. In this study, a chemoproteomic platform, termed activity-based protein profiling, was used to broadly define serine hydrolase targets in mouse brain of a panel of 29 OP and TC pesticides. Among the secondary targets identified, enzymes involved in the degradation of endocannabinoid signaling lipids, monoacylglycerol lipase, and fatty acid amide hydrolase were inhibited by several OP and TC pesticides. Blockade of these two enzymes led to elevations in brain endocannabinoid levels and dysregulated brain arachidonate metabolism. Other secondary targets include enzymes thought to also play important roles in the nervous system and unannotated proteins. This study reveals a multitude of secondary targets for OP and TC pesticides and underscores the utility of chemoproteomic platforms in gaining insights into biochemical pathways that are perturbed by these toxicants.