Background Pre-exposure prophylaxis (PrEP) use has been attributed to heightened rates of sexually transmitted infections (STIs), ostensibly due to increased condomless anal sex (CAS) and greater frequency of STI testing. Few qualitative studies have assessed how gay, bisexual and queer men (GBQM) who use PrEP perceive STIs and how these attitudes have evolved post-PrEP uptake. We investigated the perspectives of current and former PrEP users on STIs. Methods Annual, in-depth longitudinal interviews were conducted with 38 current and former PrEP users in Ontario (n = 18) and British Columbia (n = 20), Canada, as part of a mixed-methods implementation science study (2020–2022). Over 3 years, 109 interviews were conducted. Data analysis included reflexive thematic coding and longitudinal recurrent cross-sectional analysis using NVivo 12. Results Four STI-related themes emerged: (1) lack of STI-related concerns due to treatment and prevention optimism, (2) stigma-related concerns, (3) perceived risk among other PrEP users due to increased CAS, and (4) inconsistent testing concerns among non-PrEP users. Over time, some STI-related anxieties decreased with increased knowledge and reduced stigma. However, concerns persisted for other participants due to perceived risky sexual behaviours among other PrEP users and non-PrEP users. Both current and former PrEP users who expressed STI-related apprehensions consistently indicated adopting risk-reduction strategies, including condom use and having fewer sexual partners throughout the study. Conclusions Findings show how varied STI perceptions and experiences among current and former PrEP users shaped sexual decision-making over time. Providers, public health experts, and policymakers should develop a more comprehensive strategy to address STI concerns among GBQM.