Although its usage is partially banned in developed countries, organophosphate (OP) pesticide diazinon finds extensive agricultural application in our country (Turkey). This study was conducted to evaluate the effects of diazinon on total glutathione (tGSH), GSH-related enzymes, cholinesterase (ChE) enzyme activities, and lipid peroxidation in the liver of Oreochromis niloticus, a freshwater fish, as a model organism. Fish were exposed to 0.1, 1, and 2 mg/L sublethal concentrations of diazinon for 1, 7, 15, and 30 days. Total GSH levels, GSH-related enzyme and ChE-specific activities, and malondialdehyde (MDA) levels were analyzed using spectrophotometric methods. tGSH levels are decreased at 1 day, while they were increased in the long-term period. GSH-related enzyme activities are affected by diazinon exposure, except glutathione reductase (GR; EC 220.127.116.11). Diazinon displayed an oxidative stress-inducing potential and it increased lipid peroxidation. Similar inhibition levels were observed in acetylcholinesterase (AChE; EC 18.104.22.168) and butyrylcholinesterase (BChE; EC 22.214.171.124.) enzyme activities, and these inhibitions were not dose dependent. ChE inhibition-related oxidative stress was observed using its correlation with elevated tGSH levels and increased glutathione S-transferase (GST; EC 126.96.36.199) enzyme activities; that reflects the diazinon-induced oxidative stress in the liver of O. niloticus. According to the results of the present study, tGSH level and GST-specific activity are suitable for reflecting the toxic effects of diazinon in fish.