The goal of this study was to investigate the association between serum oestradiol levels and clinically significant erectile dysfunction in a cohort of men presenting for andrological evaluation. Retrospective review was conducted of patients that presented to a urologist with practice in andrology over an 18‐month period. Patients completed the Male Sexual Health Questionnaire and had serum total testosterone and oestradiol measurements prior to 10:30 a.m. via immunoassay. t Tests, chi‐square tests and multivariate logistic regression were used to compare clinical characteristics between those with adequate erectile function (erection scale score > 2) vs. clinically significant erectile dysfunction (erection scale score ≤ 2). Among 256 patients, average age was 49 years (SD 15), average serum oestradiol was 22.3 pg/ml (SD 10.6), and average serum total testosterone was 465.9 pg/ml (SD 206.3). On multivariate logistic regression, serum oestradiol was associated with clinically significant erectile dysfunction (OR 1.52 per SD increase, 95% CI 1.11–2.09, p = 0.009) when controlling for serum total testosterone, age, body mass index and smoking status. These results warrant future studies on the utility of measuring serum oestradiol in patients with erectile dysfunction and the use of aromatase inhibitors in patients with erectile dysfunction and elevated serum oestradiol.