Women who have sex with women in Southern Africa, where HIV prevalence is high, are often presumed to have minimal risk for sexually transmitted infections (STI) and HIV despite research documenting female-to-female transmission. This study examined the demographic and social factors contributing to female-to-female STI/HIV transmission knowledge among Southern African women who have sex with women using an integrated model of health literacy. In collaboration with community-based organisations in Botswana, Namibia, South Africa and Zimbabwe, data were collected through anonymous surveys (N = 591). Multivariable stepwise forward logistic regression assessed independent associations between participant characteristics and high vs. low knowledge using five items. Overall, 64.4% (n = 362) of women had high knowledge; 35.6% (n = 200) had low knowledge. Higher education (adjusted odds ratio [aOR]: 2.24, 95% confidence interval [CI]: 1.48, 3.40), regular income (aOR: 2.14, 95% CI: 1.43, 3.21), residence in Botswana (aOR: 3.12, 95% CI: 1.15, 8.48) and having ever received tailored STI/HIV information (aOR: 2.17, 95% CI: 1.41, 3.32) predicted significantly higher odds of high knowledge in the final multivariable model. Results suggest opportunities for peer-led sexual health programming and expanded HIV prevention campaigns addressing women who have sex with women.