Our aim was to assess the impact of the association between elevated oestradiol (E-2) and low testosterone (T) levels on erectile dysfunction (ED) severity. A total of 614 male patients with ED and a normal or low T level in association with normal or elevated E-2 levels were enrolled. Patients underwent routine laboratory investigations in addition to measurements of total T, total E-2, follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin. We compared the responses to the erectile function domain, Q3 (achieving erection) and Q4 (maintaining erection) of the International Index for Erectile Function (IIEF) score in patients with the following: normal T and E-2 levels; low T level; low T level and elevated E-2 level; and elevated E-2 level. Of the patients included, 449 (73.1%) had normal T and E-2 levels, 110 (17.9%) had a low T level, 36 (5.9%) had a low T level and an elevated E-2 level, and 19 (3.1%) had an elevated E-2 level. Increased ED severity was significantly associated with low T levels, elevated E-2 levels, and both a low T level and an elevated E-2 level. Additionally, the mean values of the EF-domain, Q3 and Q4 were significantly lower in patients with both a low T level and an elevated E-2 level compared to patients with any condition alone. In conclusion, a low T level had the primary effect on erectile function; however, a concomitantly elevated E-2 level had an additive impairment effect.