Considering that diesel oil is one of the most common aquatic contaminants, we compare the oxidative stress between two species of fish with different habitats (Pterygoplichthys anisitsi, benthic and Oreochromis niloticus, nektonic) exposed to diesel oil. Malondialdehyde concentrations (MDA) and the activities of ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase were all analyzed in the fishes' livers and gills after 2 and 7 days of exposure to different concentrations of diesel (0.1 and 0.5 mL/L). In the tilapia, MDA levels and the activities of EROD and GST activity in the liver, as well as MDA levels and the activities of GST and SOD in the gill had statistically significant differences between the treatments and between the times of exposure. For the catfish, the same occurred in the case of MDA, EROD, and SOD in the liver and in CAT and SOD in the gills. There were significant differences in the enzyme activity and lipid peroxidation between the species. Although the activity of most enzymes seemed to be more expressive and responsive to diesel in O. niloticus, diesel oil also caused significant effects on oxidative stress parameters in P. anisitsi, even though this species is benthic and thus has less access to insoluble fractions of diesel oil. Therefore, both species can be used as sentinel organisms in environmental biomonitoring of diesel contamination.