In Tunisia, the Mediterranean fruit fly (or medfly), Ceratitis capitata (Wiedemann), is one of the most damaging insect pests in horticulture. It is primarily managed with the application of organophosphate insecticides, notably malathion. In this work, we investigate the toxicological and biochemical effects of malathion application on C. capitata adults from the Gafsa oasis in south Tunisia. The toxicological study was carried out by a contact test, and the biochemical activities were analyzed via acetylcholinesterase (AChE) activity, as well as by using catalase (CAT), glutathione S- transferase (GST) and malondialdehyde (MDA) levels as oxidative stress biomarkers in whole bodies of adult Mediterranean fruit flies. For toxicological effects, the highest concentrations (50 ppm) generated 66.66% of mortality after 24 h of exposure, whereas the lowest concentration (3.125 ppm) achieved 30% of mortality for the same duration. For biochemical activities, malathion application increased MDA content. In addition, results indicated that malathion caused decreased AChE activity, decreased CAT activity, and increased GST activity compared to controls (non-treated flies). We infer that while malathion resistance in C. capitata populations from Gafsa oasis has not yet been described, a marked increase in Glutathione S-transferase levels after exposure of Medfly adults compared to the controls strongly suggests that the oasis' Medfly populations tend to acquire resistance to malathion. Therefore, safer control alternatives should be implemented.