This study aimed to investigate the effects of diazinon on fish, focusing on hepatotoxic biomarkers and the potential protective effects of silymarin supplementation. One hundred eighty rainbow trout were randomly assigned to four groups: control, diazinon exposed (0.1 mg L−1), silymarin supplemented (400 mg kg−1), and diazinon + silymarin. Blood samples and liver tissue were collected after 7, 14, and 21 days of exposure to analyze biochemical parameters and oxidative biomarkers. Diazinon exposure in fish resulted in liver damage, as indicated by increased antioxidant enzyme activities in the hepatocytes. Silymarin showed the potential to mitigate this damage by reducing oxidative stress and restoring enzyme activities. Nevertheless, diazinon increased creatine phosphokinase activity, which may not be normalized by silymarin. Exposure to diazinon increased glucose, triglyceride, and cholesterol levels, whereas total protein, albumin, and globulin levels were significantly decreased in fish. However, silymarin controlled and maintained these levels within the normal range. Diazinon increased creatinine, urea, uric acid, and ammonia contents. Silymarin could regulate creatinine, urea, and uric acid levels while having limited effectiveness on ammonia excretion. Furthermore, diazinon increased malondialdehyde in hepatocytes, whereas administration of silymarin could restore normal malondialdehyde levels. Overall, silymarin showed potential as a therapeutic treatment for mitigating oxidative damage induced by diazinon in fish, but its effectiveness on creatine phosphokinase, glutathione reductase, and ammonia may be limited.