This study examined the status of oxidative stress in 599 couples undertaking in vitro fertilization (IVF) treatment and its association with reproductive hormones, smoking, and outcomes. Oxidative stress biomarkers such as malondialdehyde (MDA), 8-hydroxy-2-deoxyguanosine (8-OHdG), hydrogen peroxide (H2O2), catalase (CAT), and total antioxidant capacity (TAC) were determined in follicular fluid and seminal plasma. Tail moment (TM) was used to evaluate DNA damage in sperm and granulosa cells. Reproductive hormones in serum and cotinine (COT) in urine, follicular fluid, and seminal plasma samples were determined. We used log-binomial multivariate regression to estimate relative risks for the association between oxidative stress/DNA damage and IVF binary outcomes (fertilization rate, biochemical pregnancy, clinical pregnancy, and live birth). We observed an increase in the oxidative stress markers MDA, 8-OHdG, and H2O2 in follicular fluid and seminal plasma, but a decrease in the antioxidant protection markers CAT and TAC. The MDA, 8-OHdG, and H2O2 levels were significantly higher in seminal plasma than in follicular fluid, while TAC, CAT, and TM were higher in follicular fluid (p < 0.001). Although women were nonsmokers, COT levels >50 µg/l were observed in 5.7% (urine) and 1.4% (follicular fluid). An increase in the CAT levels of follicular fluid was associated with a 48 and 41% decrease in the risk of poor fertilization rate (≤50%) and unsuccessful live birth, respectively. After the models were adjusted for hormonal factors, the associations remained the same, except that elevated TAC in follicular fluid became significantly associated with a decrease of 42% in the risk of poor fertilization rate (≤50%).