Botanical supplements are widely used and contain diverse ingredients, including isoflavones. Food-based isoflavones have been associated with reduced breast cancer risk. However, no study has comprehensively evaluated supplements identified by isoflavone content and breast cancer risk. Associations between ever use of 28 isoflavone supplements and breast cancer risk in Ontario, Canada were evaluated using cases (n = 3,101) identified in 2002-2003 from the Ontario Cancer Registry and controls (n = 3,471) identified through random digit dialing methods. Multivariate logistic regression was used to estimate age-adjusted odds ratio (AOR) and 95% confidence intervals (CI). Several individual supplements were associated with reduced breast cancer risk (e.g., Natural HRT; AOR = 0.39; 95% CI: 0.22, 0.69; n(users) = 58). Use of any isoflavone supplements was associated with reduced risk when ≥ 3 were ever used (AOR = 0.68; 95% CI: 0.54, 0.86; n(users) = 332; p(trend) = 0.008) or any was taken >5 years (AOR = 0.75; 95% CI: 0.60, 0.94; n(users) = 325; p(trend) = 0.01); high content supplements were consistently associated with reduced risk. Risk reduction was confined to postmenopausal breast cancer for both individual and combined supplements, and was strongest in the latter among high content users who ever took ≥ 3 supplements (AOR = 0.55; 95% CI: 0.38, 0.81; n(users) = 118; p(trend) = 0.04) or any >5 years (AOR = 0.47; 95% CI: 0.27, 0.81; n(users) = 60; p(trend) = 0.03). Associations did not differ by estrogen-progesterone tumor receptor status. In conclusion, isoflavone supplements were associated with decreased postmenopausal breast cancer risk. Further research to examine these novel findings is warranted, given the low supplement use and potential limitations of our results.