The toxic effect of sublethal concentrations (1, 17.5 and 35 mg L −1 ) of pure glyphosate was evaluated on acetylcholinesterase (AChE) activity in the fish species, Cnesterodon decemmaculatus . Acute bioassays (96 h) under laboratory conditions were conducted and homogenates for each specimen corresponding to the anterior, middle and posterior body sections were performed. Fish survival was 100%, even at the highest concentration tested (35 mg L −1 ), in accordance with the low lethal toxicity reported for glyphosate. However, a significant inhibitory effect on AChE activity was recorded even for the lowest herbicide concentration tested (1 mg L −1 ), in the homogenates corresponding to the anterior body section. The inhibition ranged from 23 to 36%. The analytical determination of glyphosate in assay media by ion chromatography, was used to verify its stability. These results indicate that AChE—a neurotoxicity biomarker—in C . decemmaculatus may be affected by exposure to environmentally relevant concentrations of glyphosate.