We assessed HIV prevalence and associated sexual behaviour risks among male regular partners (MRPs) of female sex workers (FSWs) attending a peri-urban clinic in Kampala, Uganda. Between June 2012 and June 2017, we conducted a mixed-methods cross-sectional study among MRPs (aged ≥ 18 years old). Eligible and interested men were consented and offered HIV counselling and testing. Data on socio-demographic, sexual behaviour and clinical characteristics were collected. Multivariable Poisson regression with robust variance was used to identify factors associated with HIV prevalence. In-depth interviews were conducted to elicit information on participants’ knowledge of HIV and its prevention as well as sexual risk practices. Qualitative data were analysed using a thematic content approach. One hundred and sixty-two men were included in the analysis; mean age was 35 years (standard deviation ± 8.43), and most (73%) reported not using a condom during the last sexual intercourse. Overall HIV prevalence was 65 of 162 (40%). Absence of sexually transmitted infection symptoms in the last 12 months adjusted prevalence ratio (aPR) = 0.56, 95% confidence interval (CI) = [0.37, 0.85] and condom use at last sexual intercourse (aPR = 0.63, 95% CI = [0.45, 0.88]) were associated with a lower HIV prevalence. Being uncircumcised (aPR = 1.83, 95% CI = [1.22, 2.74]) and having ≥2 sexual partners in the last 12 months (aPR = 1.91, 95% CI = [1.27, 2.85]) were associated with higher HIV prevalence. Qualitative data showed that most participants reported not consistently using condoms with their FSW partners because of the emotional connections, desire to have children and trust. HIV prevalence among MRPs was high compared to the national average. HIV prevalence among MRPs was associated with high-risk sexual behaviour and circumcision status. Interventions are needed to address the high sexual behaviour risks among MRPs of FSWs as well as supporting them to access safe voluntary medical male circumcision, HIV care and support services.