Objectives Male and transgender women sex workers (TSWs) are vulnerable for sexually transmitted infections (STIs) and human immunodeficiency virus (HIV), and substance use might be a relevant contributing factor. We assessed sociodemographic characteristics and substance use among sex workers; divided into 3 groups: Transgender women sex workers, male sex workers who have sex with men only or also with females (MSW-M), male sex workers who have sex with females only (MSW-F). Methods A survey on substance use and sexual (risk) behavior was offered during routine STI screening at the Prostitution and Health Centre (P&G292) in Amsterdam. Bacterial STI positivity (chlamydia (including lymphogranuloma venereum), gonorrhea, and/or infectious syphilis), and substance use were compared (χ 2 test, Fisher exact test). Results From 2014 until 2015, 99 (60.4%) of 164 eligible visitors participated (n = 69 MSW-M [69.7%], n = 15 TSW [15.2%], and n = 15 MSW-F [15.2%]). Transgender women sex workers reported the highest number of sex partners in the previous 6 months (median: MSW-M 60 vs. TSW 300 vs. MSW-F 12; P < 0.001). The 3 groups did not differ in having condomless anal or oral sex. Bacterial STI positivity was 29.0% in MSW-M, 26.7% in TSW, and 13.3% in MSW-F ( P = 0.56). Three new HIV infections were diagnosed, all in MSW-M, whereas 20.3% of MSW-M and 20.0% of TSW were known HIV-positive compared with none of MSW-F ( P = 0.14). Illicit substance use during working time in <6 months was 40.5% among MSW-M, 40.0% among TSW, and 20.0% among MSW-F ( P = 0.02). The most reported reason for substance use was: “sex work becomes physically easier.” Conclusions Bacterial STI positivity and illicit substances use during work were high in all 3 sex worker groups, emphasizing the importance of combined and targeted interventions. In-depth qualitative research is needed to better understand intentions and reasons for substance use.