Despite years of preventive interventions, Mother-to-child transmission (MTCT) of HIV remains high in low- and middle-income countries. This study aimed to identify risk factors associated with MTCT of HIV in Ghana. A 1:1 unmatched case-control study was conducted among HIV-infected mothers and their exposed children (184 cases and 184 controls) using data from the 2021-2022 HIV-Positive Babies Audit by the National AIDS/STI Control Programme in Ghana. Only variables with missing values ≤ 5% were included in univariable logistic regression analysis. Variables with -values ≤ 0.20 were entered into multivariable logistic regression. Six variables were considered: marital status, employment status, mode of delivery, supervision of delivery, type of antiretroviral (ARV) prophylaxis for the baby, and duration of ARV prophylaxis. After adjustment, lack of ARV prophylaxis (AOR = 4.35, 95% CI: 2.41-7.83, < 0.001) and ARV prophylaxis for less than 12 weeks (AOR = 75.70, 95% CI: 17.18-333.62, < 0.001) significantly increased the odds of MTCT of HIV. The predictive power of the multivariable logistic regression model was 81%. Introducing systems to ensure that all HIV-exposed babies in Ghana receive at least 12 weeks of ARV prophylaxis is crucial for significantly reducing the burden of MTCT of HIV.