Abstract Background Although some evidence suggests that alcohol, substance use, and mental health issues diminish adherence to HIV Pre-Exposure Prophylaxis (PrEP) among gay, bisexual, and other men-who-have-sex-with-men (gbMSM), findings are somewhat inconsistent and have primarily derived from studies involving non-random samples. Medical chart extraction can provide unique insight by in part surmounting sampling-related limitations, as data for entire PrEP clinic populations can be examined. Our investigation entailed comprehensive chart extraction to assess the extent to which chart-reported alcohol, substance use, and mental health issues were associated with chart-reported PrEP nonadherence. Methods Data from medical charts of gbMSM at two PrEP clinics in Toronto, Canada were extracted for a retrospective 12-month period (02/2018-01/2019). Charts were reviewed for all patients who were 1) ≥ 18 years old; 2) gbMSM; 3) prescribed PrEP ≥ 3 months, and 4) not in a PrEP-related drug trial. Information regarding PrEP, alcohol, substance use, mental health, and sexual behavior was extracted. PrEP adherence was classified in terms of (1) any reported nonadherence, and (2) ‘suboptimal adherence,’ reflecting nonadherence patterns indicative of insufficient pharmacological protection from HIV. Multivariate logistic regression was employed to identify factors associated with adherence outcomes. Results Data were extracted from 4,292 clinic visits among 501 eligible patients (age: M = 39.1; duration on PrEP: M = 17.4 months; daily PrEP regimen = 93.8%). Hazardous/harmful drinking, club drug use, and mental health issues were reported among 8.8%, 22.2%, and 26.1% of patients, respectively. Any nonadherence and suboptimal adherence were reported among 37.5% and 12.4% of patients, respectively. Factors significantly associated with any nonadherence included age < 25 (AOR = 3.08, 95%CI = 1.54–6.15, p < .001), club drug use (AOR = 2.71, 95%CI = 1.65–4.47, p < .001), and condomless sex (AOR = 1.83, 95%CI = 1.19–2.83, p = .006). For suboptimal adherence, significant factors included age < 25 (AOR = 4.83, 95%CI = 2.28–10.22, p < .001), non-daily PrEP regimens (AOR = 2.94, 95%CI = 1.19–7.22, p = .019), missing PrEP appointments (AOR = 1.97, 95%CI = 1.09–3.55, p = .025), and club drug use (AOR = 1.97, 95%CI = 1.01–3.68, p = .033). Neither alcohol nor mental health issues were associated with nonadherence outcomes. Conclusions Chart-indicated suboptimal adherence was present among a small subgroup of PrEP-prescribed gbMSM. Adherence-related interventions should target gbMSM who use club drugs, are younger, experience challenges attending PrEP care, and are prescribed non-daily regimens. Offering long-acting injectable PrEP when available and feasible may also improve PrEP’s HIV-preventive impact among this population.