Ethanol toxicity is affected by both environmental and inherited features. Since oxidative stress is an important molecular mechanism for ethanol-induced cellular damage, the concomitant exposure to ethanol and pro-oxidative or antioxidant compounds can alter its toxicity. Here, we investigate the effects of exposure to ethanol and/or diphenyl diselenide, an organochalcogen with antioxidant properties, on parameters related to oxidative stress (thiobarbituric acid reactive species—TBARS—and δ-aminolevulinate dehydratase— δ-ALA-D activity) in mouse liver and brain. In addition, the in vitro effects of ethanol and acetaldehyde on the activity of δ-ALA-D from human erythrocytes were also investigated. Both ethanol and diphenyl diselenide decreased hepatic δ-ALA-D activity and DL dithiothreitol (DTT) reactivated this enzyme only after ethanol-induced inhibition. Moreover, ethanol increased liver TBARS levels, independently of the presence of diphenyl diselenide treatment. Brain δ-ALA-D activity and TBARS levels were not changed by ethanol or diphenyl diselenide exposure. Under in vitro conditions, acetaldehyde was a more potent inhibitor of δ-ALA-D from human erythrocytes when compared to ethanol, demonstrating a dose-dependent effect. This study indicates that hepatic δ-ALA-D is a molecular target for the damaging effect of ethanol under in vivo conditions; diphenyl diselenide and ethanol seem to inhibit δ-ALA-D by different mechanisms; acetaldehyde, a metabolite of ethanol, is probably the main molecule responsible for the inhibitory effects of the parent compound on δ-ALA-D.