Glyphosate and glufosinate ammonium are widely used herbicides in agriculture for weed elimination. These unused glyphosate and glufosinate ammonium are discharged into environmental water, soil, and even in the food chain cycle, which greatly threatens ecosystems and human health. However, the toxicological mechanism of glyphosate and glufosinate ammonium on soil non-target organisms remains largely unknown. To investigate the toxic effects of glyphosate and glufosinate ammonium on soil non-target organisms, Caenorhabditis elegans which is directly exposed to various pesticides in the soil was selected as the object of study. In this study, our results showed that the locomotion, growth, reproduction, and lethality of C. elegans exposed to different concentrations of a mixture of glyphosate and glufosinate ammonium are suppressed. Exposure to a mixture of glyphosate and glufosinate ammonium increases the production of reactive oxygen species, accelerating senescence and shortening the lifespan of C. elegans. The exposure to a mixture of glyphosate and glufosinate ammonium activates antioxidant defense responses mediated by DAF-16 and autophagy mediated by SKN-1. Our research provides a novel perspective on the mechanism underlying antioxidant defense response-mediated detoxification of pesticide residues in soil non-target organisms.