Background Amphetamine-type stimulant (ATS) use has increased in Cambodia and emerged as a significant problem among female sex workers (FSWs), potentially contributing to increased risk of HIV. We examined the prevalence of ATS use and its effect on sexual risk behaviors, and sexually transmitted infections (STI) among FSWs in Phnom Penh, Cambodia. Methods A one-year prospective study among young women engaged in sex work in brothels, entertainment establishments and on a freelance basis. Socio-demographics, sexual risks, and recent ATS use were assessed by self-report. Blood and urine samples were collected to detect HIV, Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (GC). Bivariate and multivariate longitudinal analyses were conducted to assess the effects of ATS use on number of sex partners, inconsistent condom use with paying partners and incident STI. Results ATS use was higher among women working freelance (35.6%) and in brothels (34.8%) compared to women working in entertainment establishments (17.7%) or in multiple venues (14.8%). ATS users reported more sex partners and days drunk in the previous month. In multivariate longitudinal analysis, ATS use was associated with having a higher number of sex partners (Adjusted Risk Ratio 1.49; 95% CI: 1.00–2.21) and incident STI (Adjusted Odds Ratio 5.41; 95% CI: 1.15–25.48), but not inconsistent condom use with paying partner. Conclusion ATS users had more sex partners, high level of alcohol use, and were at increased risk of STI. Our findings underscore ATS use as an important emerging risk exposure that should be integrated into HIV prevention interventions targeting this population.