Background: Female sex workers (FSWs) are at a higher risk of contracting and transmitting HIV and other sexually transmitted infections (STIs). We aimed to explore the sexual behaviour, knowledge and attitudes towards STIs, barriers, support and intervention for STI screening. Methods: In this community-based, concurrent quantitative–qualitative, exploratory mixed-methods study, we administered 87 semi-structured quantitative and six in-depth interviews to purposively select FSWs in Gulu City, Uganda. The qualitative methodology was based on descriptive phenomenology. Results: The quantitative study included 87 FSWs, with a median age of 28 years. Overall, 87.4% (n = 76) participants reported condom use during their last sexual encounter. Eighty-three (95.4%) participants were aware of their HIV status, with 6% (n = 5) reporting being HIV-positive. Seventy-six (87.4%) participants reported contracting at least one STI during sex work. In addition, 66.7% (n = 58) of the participants reported being screened for STIs in the past three months, with vulvovaginal candidiasis (55.3%, n = 42) and syphilis (32.9%, n = 25) being the most common STIs. However, only 2.3% (n = 2) of the participants reported current STIs symptoms. In the qualitative study, the main themes encompassed sexual behaviour, including condom-use negotiation strategies and challenges, as well as screening facilitators and barriers. Condom-use negotiation tactics involved leveraging health-risk information, increased charges for unprotected sex, and outright refusal. Complex challenges included limited agency in promoting safe sex due to economic reliance, vulnerability to violence, and difficulties in controlling the sexual environment. Facilitators for regular screening included social influences, emotional relief, established testing habits, and accessible clinics, while barriers encompassed cost, stigma, doubts about test accuracy, and inconvenient clinic hours. Conclusions: FSWs face significant vulnerabilities in Gulu City, Uganda, regarding STIs. While encouraging trends such as high HIV awareness and condom usage were observed, persistent challenges in STI screening, negotiation of safe sex practices, and access to prevention and treatment remain evident. To address these concerns, targeted interventions should be developed to enhance STI screening accessibility, empower sex workers with effective negotiation skills, and provide comprehensive support for STI prevention and treatment, thereby contributing to improved overall sexual health and well-being among this marginalized population.