By surveying extensive epidemiological behavioural and sexual risk factors in a Korean twin cohort, risk factors for human papillomavirus (HPV) infection were investigated in South Korea. A total of 912 vaginal specimens were collected from the Healthy Twin Study, consisting of twins and their families. A range of epidemiological, behavioural, and sexual activity characteristics were evaluated using multivariate logistic regression analyses of family and twin relationships, adjusted to elucidate the risk factors for HPV infection. Of the various epidemiological characteristics, the possibility of extramarital affairs [odds ratio (OR) 2·48, 95% confidence interval (CI) 1·02–6·02] significantly increased the prevalence of HPV infection. Our multivariate regression analysis indicated that oral contraceptive use (OR 40·64, 95% CI 0·99–1670·7) and history of sexually transmitted disease (OR 2·56, 95% CI 0·93–7·10) were strongly associated with an increase in HPV infection. On the other hand, more frequent vaginal douching (OR 0·32, 95% CI 0·13–0·77) significantly decreased the prevalence of HPV infection. Our results suggested that HPV infection is associated with both biological and behavioural factors.