Objectives The burden of HIV and other sexually transmitted infections (STIs) remains high in sex workers globally, calling for strengthening targeted prevention strategies, including HIV pre-exposure prophylaxis (PrEP). The study’s objective was to assess HIV and STI burden among female, male and transgender sex workers in Flanders, Belgium, to guide targeting of PrEP strategies for sex workers. Methods We conducted a retrospective analysis of routine data collected between January 2016 and December 2019 by community-based organisations providing sexual healthcare services for sex workers in Flanders. HIV prevalence stratified by gender was assessed and associations with sociodemographic characteristics were explored using bivariable and multivariable logistic regression. Positivity rates of chlamydia, gonorrhoea and syphilis tests were used as proxy indicators for STI burden. Results The study included a total of 6028 sex workers, comprising 5617 (93.2%) female, 218 (3.6%) male and 193 (3.2%) transgender sex workers. The HIV prevalence was 0.3% among female, 8.9% among male and 12.3% among transgender sex workers. Engaging in escort sex work and originating from South America or Sub-Saharan Africa were associated with a higher likelihood of having acquired HIV. The positivity rate for gonorrhoea was higher among male sex workers (5.2% vs 2.2%) and syphilis was more frequently detected among male and transgender sex workers (3.0% and 6.1% vs 0.5%), all compared with female sex workers. Conclusions HIV combination prevention, including improved access to PrEP, should be strengthened among sex workers in Flanders, with particular attention to male and transgender sex workers.