Triflumezopyrim is a novel mesoionic pesticide used in Asian rice fields to control brown plant hopper, which can reach aquatic environments through drift during application or surface runoff. Thus, this study aimed to investigate the effect of chronic exposure of sub-lethal concentrations of triflumezopyrim (2.468 ppm, 1.480 ppm, and 0.740 ppm) on freshwater carp, Labeo rohita.This study encompassed a multi-pronged approach, including molecular docking studies to elucidate the interactions between triflumezopyrim and selected proteins, an assessment of 8-OHdG (8-hydroxy-2-deoxyguanosine) activity to gauge DNA damage, histopathological examinations to identify cellular alterations, and expression of genes involved in oxidative stress, DNA repair, and apoptosis in L. rohita. We observed dose-dependent responses in 8-OHdG activity and the expression of select genes, with higher concentrations of triflumezopyrim yielding more pronounced transcriptional alterations. Notably, histopathological examinations of liver and brain tissues vividly portrayed the impact of triflumezopyrim on L. rohita. These findings contribute to the assessment of biological toxicity and the environmental footprint left by chemical pollutants such as triflumezopyrim. The study emphasizes the crucial role of monitoring histopathological alterations, 8-OHdG activity and gene expression changes as potential biomarkers for assessing exposures to triflumezopyrim. This research provides valuable insights into the ecological implications of triflumezopyrim in aquatic ecosystems.