To prospectively compare serum hormone levels and the incidence of hormonal pathologies between men with and without erectile dysfunction, and investigate risk factors that might predict hormonal pathologies in men complaining of erectile dysfunction. The study included 262 men with erectile dysfunction and 53 healthy men with no erectile dysfunction as a control group. All men enrolled in the study were evaluated with a detailed history, physical examination, international index of erectile function (IIEF-5), and serum hormone measurement. Hypotestosteronemia was considered as serum total testosterone value of < 3 ng/mL, and hyperprolactinemia was considered as serum prolactin level of > 18 ng/mL. Serum hormone levels and the incidence of hormonal abnormalities were compared between the two groups. In addition, risk factors for hormonal abnormalities were investigated. There were no significant differences in the mean serum FSH (p = 0.212), LH (p = 0.623), testosterone (p = 0.332) and prolactin values (p = 0.351) between the men with and without erectile dysfunction. Hypotestosteronemia was detected in 29 (11%) of the erectile dysfunction group and in 2 (3.7%) of the control group, revealing no significant difference (p = 0.104). Hyperprolactinemia was detected in 25 (9.5%) of the erectile dysfunction group and in 2 (3.7%) of the control group, revealing no significant difference (p = 0.171). To investigate risk factors that might predict hormonal pathologies, there were no significant differences in the patient age, duration of the sexual dysfunction, smoking history and duration, the presence of chronic disease and the type of erectile dysfunction. Our findings suggest that hormonal measurement should not be routinely performed in the initial evaluation of men presenting with erectile dysfunction, and may be necessary based only on the findings obtained with a careful history and physical examination.