Background Before 2019, availability of affordable HIV preexposure prophylaxis (PrEP) was limited in the Netherlands. Consequently, some men who have sex with men and transgender persons obtained PrEP informally, that is, without a prescription. Methods Between September 2017 and August 2018, we enrolled informal PrEP users in a cohort study (Informal PrEP [InPrEP]) at the Amsterdam Sexually Transmitted Infections (STI) Clinic. We compared PrEP use, PrEP eligibility and intention, chemsex, and STI positivity between 3 groups: (1) informal PrEP users enrolled in InPrEP, (2) nonenrolled informal PrEP users, and (3) non-PrEP users. Also, we assessed intention and associated characteristics among non-PrEP users who were eligible for PrEP. Results During the study period, we enrolled 118 informal PrEP users in InPrEP who were compared with 224 nonenrolled informal PrEP users and 4975 non-PrEP users. Preexposure prophylaxis eligibility rates were 83.1%, 93.4%, and 63.0%; chemsex were reported by 39.1%, 48.1%, and 11.7%; and STI positivity was reported by 28.0%, 42.5%, and 20.3%, respectively. Among PrEP-eligible non-PrEP users, PrEP intention was 16.6% and associated with increasing age, non-Dutch origin, higher educational level, receptive condomless anal sex, increasing number of sex partners, chemsex, and sex with men only. Conclusions Most informal PrEP users are eligible to use PrEP and have a clear sexual health care need. Especially in settings with limited PrEP access, providing the necessary care for informal PrEP users is important. The intention to use PrEP is low among PrEP-eligible STI clinic visitors. Further research should investigate modes to increase PrEP intention among PrEP-eligible persons.