BACKGROUND: Cambodia has been well recognized for its success in the fight against the HIV epidemic. However, challenges remain in eliminating HIV infections in key populations, including women working in entertainment establishments, such as massage parlors, karaoke bars, or beer gardens. This study explored the prevalence of HIV and identified factors associated with HIV infection among female entertainment workers (FEWs) in Cambodia. METHODS: This national biological and behavioral survey was conducted in 2016 in Phnom Penh and 17 provinces. We used a two-stage cluster sampling method to recruit FEWs for HIV testing performed on-site and face-to-face interviews using a structured questionnaire. We investigated factors associated with HIV infection using multiple logistic regression. RESULTS: This study included 3149 FEWs with a mean age of 26.2 years (SD 5.7). The adjusted prevalence of HIV was 3.2% (95% CI 1.76-5.75). In the multiple logistic regression model, the odds of HIV infection were significantly higher among FEWs in the age group of 31 to 35 (AOR 2.72, 95% CI 1.36-8.25) and 36 or older (AOR 3.62, 95% CI 1.89-10.55); FEWs who were not married but living with a sexual partner (AOR 3.00, 95% CI 1.16-7.79); FEWs who had at least ten years of formal education (AOR 0.32, 95% CI 0.17-0.83); FEWs who reported having abnormal vaginal discharge (AOR 3.51, 95% CI 1.12-9.01), genital ulcers or sores (AOR 2.06, 95% CI 1.09-3.17), and genital warts (AOR 2.89, 95% CI 1.44-6.33) in the past three months; and FEWs who reported using illicit drugs (AOR 3.28, 95% CI 1.20-4.27) than their respective reference group. The odds of HIV infection were significantly lower among FEWs working in karaoke bars (AOR 0.26, 95% CI 0.14-0.50) and beer gardens (AOR 0.17, 95% CI 0.09-0.54) than among freelance FEWs. CONCLUSIONS: The prevalence of HIV among FEWs in Cambodia remains much higher than that in the general population. These findings indicate that differentiated strategies to address HIV and other sexually transmitted infections should be geared towards FEWs working as freelancers or in veiled entertainment venues such as massage parlors and freelance sex workers. Prevention efforts among venue-based FEWs should be sustained.