There are several types of pesticides to control pests and several new types coming into use that could be less toxic compared to the old ones. Pesticide-induced oxidative stress, which is one of the main mechanisms of toxicity, is the research area focused most on over the last decade. There are several different studies in the literature on whether pesticide exposure induces oxidative stress parameter-mediated toxicity. Pesticide-induced oxidative stress level depends on the biochemical features of mammalian systems. Imidacloprid is a neonicotinoid pesticide in wide use that is considered safe; however, it has been reported in different studies that it may cause changes in oxidative stress parameters. We investigated the dose- and time-dependent effects of imidacloprid on acetylcholinesterase (AChE), lactate dehydrogenase (LDH), and glutathione (GSH) levels in the L-929 fibroblast cell line. The effects of 1-500 μg imidacloprid dose range on AChE, GSH, and LDH were investigated. LDH levels were significantly increased dose dependently in the 250 and 500 ng imidacloprid groups compared to the control group. GSH levels nonsignificantly decreased dose dependently and GSH levels were lower in the 500 ng imidacloprid group compared to the control group. There were no significant differences between the groups in AChE levels. These results indicated that high doses of imidacloprid may induce oxidative stress in fibroblast cells.