We assess the relative contribution of viral and bacterial sexually transmitted infections (STIs) on HIV acquisition among southern African women in a nested case-control study within the Methods for Improving Reproductive Health in Africa (MIRA) trial. Cases were women with incident HIV infection; controls were HIV-uninfected at the time of case seroconversion selected in a 1 to 3 case to control ratio (risk-set sampling), matched on study site and time of follow-up. Conditional logistic regression models were used to calculate adjusted odds ratios (AORs) and population-attributable fractions (PAF). Among 4948 enrolled women, we analysed 309 cases and 927 controls. The overall HIV incidence rate was 4.0 per 100 women-years. The incidence of HIV infection was markedly higher in women who had prevalent Herpes simplex virus type 2 (HSV-2) (AOR: 2.14; 95% confidence interval [CI]: 1.55-2.96), incident HSV-2 (AOR: 4.43; 95% CI: 1.77-11.05) and incident Neisseria gonorrhoeae (AOR: 6.92; 95% CI: 3.01-15.90). The adjusted PAF of HIV incidence for prevalent HSV-2 was 29.0% (95% CI: 16.8-39.3), for incident HSV-2 2.1% (95% CI: 0.6-3.6) and for incident N. gonorrhoeae 4.1% (95% CI: 2.5-5.8). Women's greatest risk factors for HIV acquisition were incident bacterial and viral STIs. Women-centred interventions aimed at decreasing HIV incidence in young African women need to address these common co-morbid conditions.