This study aimed to (1) estimate the prevalence of illicit drug use in night-time entertainment districts across five major cities in Australia; and (2) validate self-reported drug use using biochemical marker oral swabs.Street intercept surveys and oral drug swabs conducted over a 7-month period during 2011-12.The night-time entertainment districts of three metropolitan cities (Sydney, Melbourne and Perth) and two regional cities (Wollongong and Geelong) in Australia, between the hours of 10 p.m. and 5 a.m.A total of 7581 individuals agreed to participate in the survey (93% response rate). More than half (62%) the sample was male, with a median age of 22 years (range 18-73).Patrons were approached in thoroughfares and while entering and leaving licensed venues. Data collected included demographics and current session alcohol and other substance use. Drug swabs (n = 401) were performed with a subsample of participants.Approximately 9% [95% confidence interval (CI) = 7-12%] of participants self-reported consumption of illicit or non-prescribed pharmaceutical drugs prior to interview; of those, 81% identified psychostimulants as the drug used. One in five drug swabs returned a positive result, with psychostimulants the most commonly detected drugs (15%; 95% CI = 12-19%). Kappa statistics indicate agreement between self-report of any illicit drug and a positive drug swab is in the slight range [κ = 0.12 (95% CI = 0.05-0.20) P = 0.000].Self-report findings suggest drug use in Australian nightlife is common, although still very much a minority past-time. Drug swabs indicate a higher prevalence of use (20%) than self-report (9%), which suggests that self-reported drug use may not be reliable in this context.