Data about the impacts of hemodialysis on antioxidant status and markers of oxidative stress are controversial, probably due to the use of different methodological approaches. The aim of this study was to assess the changes in the oxidative damage markers and antioxidant enzymes, and the serum antioxidant capacity by using in vitro model systems of free radical generation before and after one hemodialysis session. Blood samples were collected from 40 patients with kidney failure before and after hemodialysis. In pre- and post-hemodialysis serum samples, concentrations of biomarkers of oxidative damage and the activities of antioxidant enzymes were measured, as well as the in vitro antioxidant potential. The high concentrations of oxidative stress markers in serum of kidney failure patients were decreased after one hemodialysis session. In pre-hemodialysis, low activities of antioxidant enzymes were observed, including paraoxonase-1, however paraoxonase-1 activity was partially recovered after hemodialysis. Crocin bleaching and radical scavenging assays showed that serum antioxidant potential was decreased after hemodialysis. Although one hemodialysis session increased paraoxonase-1 activity and decreased oxidative stress markers, it caused a decrease in the serum antioxidant potential. Future research is needed to prospect strategies to mitigate the impacts of oxidative stress in the scenario of hemodialysis repetitions.