BACKGROUND:This study examined the criterion validity of the online Active Australia Survey, using accelerometry as the criterion, and whether self-report bias was related to level of activity, age, sex, education, body mass index and health-related quality of life. METHODS:The online Active Australia Survey was validated against the GENEActiv accelerometer as a direct measure of activity. Participants (n = 344) wore an accelerometer for 7 days, completed the Active Australia Survey, and reported their health and demographic characteristics. A Spearman's rank coefficient examined the association between minutes of moderate-to-vigorous physical activity recorded on the Active Australia Survey and GENEActiv accelerometer. A Bland-Altman plot illustrated self-report bias (the difference between methods). Linear mixed effects modelling was used to examine whether participant factors predicted self-report bias. RESULTS:The association between moderate-to-vigorous physical activity reported on the online Active Australia Survey and accelerometer was significant (rs = .27, p < .001). Participants reported 4 fewer minutes per day on the Active Australia Survey than was recorded by accelerometry (95% limits of agreement -104 - 96 min) but the difference was not significant (t(343) = -1.40, p = .16). Self-report bias was negatively associated with minutes of accelerometer-recorded moderate-to-vigorous physical activity and positively associated with mental health-related quality of life. CONCLUSIONS:The online Active Australia Survey showed limited criterion validity against accelerometry. Self-report bias was related to activity level and mental health-related quality of life. Caution is recommended when interpreting studies using the online Active Australia Survey.