This study aimed to assess the water quality of the Perequê River, Porto Belo, Santa Catarina, Brazil, through a biomarker approach in the native fish species Geophagus brasiliensis, and the sediment chemical quantification of the herbicides glyphosate and bentazone used in irrigated rice plantations. This river is used for the public water supply of two municipalities. The first sampling (S1) was in November 2016, in the spring season and the second (S2), in March 2017, in the fall season. In each sampling, two points of the river were analyzed, one upstream of the accumulation dam and the water catchment point for water supply of the Porto Belo WTS (P1), and another downstream (P2) with water, sediment, and fish sampling. Biotransformation, oxidative stress, histopathological and genotoxic biomarkers were analyzed in different tissues. The presence of glyphosate was detected in the sediment (11.7 µg·kg-1) from the upstream point of the water catchment site (P1) in spring. The lower activity of the antioxidant enzyme superoxide dismutase (SOD), as well as the increased damage to renal DNA and hepatic tissue, coincided with the lower muscular and cerebral acetylcholinesterase activities (AChE) at P1, in relation to P2 in the spring season, with a lower rainfall index (81.8 mm3). A seasonal variation was also observed between the spring and fall seasons, in fish responses to biomarkers. Reduction of muscle AChE activity and biotransformation enzymes ethoxyresorufin-O-deethylase and glutathione S-transferase and antioxidant enzymes such as, SOD and glutathione peroxidase, as well as increased brain DNA damage, coincided with the highest number of tissue lesions in the liver and gills in the spring, regardless of the sampling point. The results suggested that the Perequê River is contaminated by xenobiotics and probably herbicides from irrigated rice plantations, indicating damages to the biota and a potential public health risk.