Unconventional and uncontrolled use of agricultural pesticides and their influence in aquatic ecosystems during drainage process causes the accumulation of these toxins in body tissues of fish, and finally, it endangers human health. In order to determine the amount of pollution of Roundup pesticide in aquatics, acute and chronic effects of this poison on gill, kidney, and liver tissues and biochemical activities of cerebral acetyl cholinesterase and liver catalase in rainbow trout (Oncorhynchus mykiss) were investigated. To determine LC 50 of Roundup pesticide in rainbow trout, acute doses of Roundup were introduced to fish tanks and fish mortality was recorded for 96 h, and Roundup LC 50 was determined using SPSS Probit test. Chronic doses were determined based on the obtained LC 50 , and the effects of these concentrations were assessed on gill, kidney and liver tissues and cerebral acetyl cholinesterase and liver catalase activities over 28 days. Based on histopathology results, the following changes were observed: adhesion of secondary lamellae, bending of secondary lamellae in gill tissue, glomerular wrinkling, dilatation of Bowman's capsule space in kidney tissue and necrosis, cellular swelling, and lipid degeneration in liver tissue. Cerebral acetyl cholinesterase and liver catalase activities significantly reduced in groups exposed to Roundup herbicide compared to the control group (p < 0.05). Generally, chronic concentrations of Roundup herbicide cause undesirable tissue and enzymatic changes in antioxidant system of rainbow trout. Therefore, assessment of biochemical factors and histopathological studies can be used as biomarkers in tracing the effects of agricultural toxins on aquatic habitat.