Background Female infertility is a public health problem, contributing to 65.9% of infertility in Tanzania. Lifestyle and oxidative stress (OS) have been suggested to affect female fertility; however, there are limited and controversial reports. This study aimed to determine the lifestyle and OS status of fertile and infertile women and relate lifestyle and OS to female infertility. The study was conducted among randomly selected 48 fertile and 48 infertile women aged 15–49 years attending a gynaecological clinic at Muhimbili National Hospital. Lifestyle information was collected using a proforma, and a sandwich ELISA method was used to analyse OS biomarkers (malondialdehyde and total antioxidant capacity). Chi-square, independent t test and correlation coefficient were used for data analysis at p < 0.05. Results Infertile women were significantly older (33.4 versus 30.3 years, p = 0.03), got married at later ages (25.0 versus 22.8 years, p = 0.02), were in employment (62.5% versus 29.2%, p = 0.001), had attained university degree (56.3% versus 27.1%, p = 0.004) and were more obese, more likely to consume alcoholic drinks (p = 0.02) and less likely to use supplements (p = 0.000) than fertile women. Infertile women had statistically significant higher malondialdehyde (p = 0.000) and lower total antioxidant capacity levels (p = 0.000) than fertile women. The total antioxidant capacity level had a statistically significant negative correlation with alcohol consumption (r = −0.27, p = 0.008) and obesity (r = −0.32, p = 0.002) and a positive correlation with supplement use (r = 0.24, p = 0.02). Also, malondialdehyde level had a statistically significant positive correlation with alcohol consumption (r = 0.31, p = 0.002) and obesity (r = 0.28, p = 0.007). Conclusions The differences in socio-demographic characteristics, lifestyle and oxidative stress between fertile and infertile women may account for difficulties in achieving pregnancy among infertile women in Tanzania. Lifestyle factors significantly affect oxidative stress. Health education on appropriate lifestyles that support female fertility and emphasis on supplement use during the preconception period may be beneficial in the management of female infertility.