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HIV incidence remains alarmingly high among female sex workers (FSWs) in Uganda, necessitating targeted interventions. This study aimed to identify individual and provider-level barriers and facilitators to primary HIV prevention among FSWs in an urban setting. Focus groups involving FSWs and healthcare providers (HCPs) were conducted to inform the development of tailored prevention interventions. Results revealed that all participants had mobile phones, recent sexual activity, and a history of HIV testing, with high rates of sexually transmitted infections and varying HIV test results. FSWs displayed a well-informed awareness of HIV transmission risks, emphasizing the threat for those not testing positive. They unanimously recognized the crucial role of HIV testing in informing, mitigating risks, promoting ART use, and endorsing consistent condom usage. Despite heightened awareness, HCPs noted potential underestimation of vulnerability. Various challenges, including inadequate condom usage, substance abuse, and client dynamics, underscored the complexity of safeguarding against HIV transmission among FSWs. Widespread alcohol and drug use, including marijuana, kuber, and khat, served as coping mechanisms and social facilitators. Some FSWs successfully reduced alcohol intake, highlighting challenges in addressing substance use. FSWs preferred group discussions in health education programs, emphasizing peer interactions and the effectiveness of visual aids in HIV prevention education. This study provides comprehensive insights to guide the development of targeted interventions addressing the multi-faceted challenges FSWs face in HIV prevention.