BackgroundThis study investigated athlete knowledge of the World Anti-doping Agency (WADA) Prohibited Substances List and the effects of four well-known performance enhancing substances (PES).MethodsA sample of 1925 elite and sub-elite athletes (mean age 20.6 years) completed a questionnaire about the banned status of 30 substances/methods and their knowledge of the effects of amphetamines, anabolic steroids, growth hormone and erythropoietin.ResultsAthletes showed limited understanding of the WADA Prohibited Substances List, scoring 32.2% correct, 36.3% incorrect, and 31.4% indicated they did not know the status of 30 substances. Responses of >50% correct were given for only eight substances/method: anabolic steroids, amphetamines, blood doping, erythropoietin, caffeine, vitamins/minerals, protein powders and iron. Athletes demonstrated moderate knowledge of the desired effects of the four PES (49% correct), but poor knowledge of their adverse effects (29% correct). Age, sex, ethnicity, professional/amateur status, and current competition level were significant predictors of the number of correct responses (r2 = 0.16, p < 0.05). Athletes most likely to provide correct responses were male, 19–22 year-olds, Caucasian, professional and international representatives.ConclusionThis comprehensive study of anti-doping demonstrated that Australian athletes had limited knowledge of a wide range of substances and PES. Better targeted drug education towards younger and non-professional athletes and evaluation of current anti-doping programs are warranted.