Objective To investigate the biological effects of the Mucuna pruriens (M. pruriens) seed extracts that lacked L-DOPA, which was formerly reported as the active ingredient, on erectile dysfunction (ED) both in vitro and in vivo. Methods Seed of M. pruriens plant that cultivated in Mae Taeng District, Chiang Mai Province, Thailand, was collected. Component of its seeds were extracted and isolated into 2 fractions using methanol, polar and nonpolar. Each fraction was investigated for phytochemicals using gas chromatography and mass spectroscopy and was screened for biological activity in vitro using three different cell lines. The most biological active fraction was used to treat both streptozotocin (STZ)-induced diabetes mellitus-erectile dysfunction (DM-ED) male Wistar rats and normal rats (n = 6 per groups) to compare the effect on sexual behavior parameters, including number of intromission, mounting and ejaculation, with that of rats given Sildenafil by individually pairing with their female counterparts. Penile tissues and serums were collected to determine histological structure, related gene expression and biomolecules. Results The phytochemicals of the polar fraction were possibly catechol and its derivatives plus polyphenols, whereas the nonpolar fraction consisted of lipid derivatives. L-DOPA was not detected in either of the extracts. The polar fraction was able to up-regulate the expression of ED-related genes including eNOS and nNOS in vitro which subsequently promotes nitric oxide production and maintains intracellular cyclic guanosine monophosphate levels. When administrated to DM-ED rats, the polar extract significantly improved all sexual behavior parameters in DM-ED rats compared to untreated group (18.3 ± 1.8 to 10.8 ± 2.9 for intromission, 9.8 ± 2.2 to 5.7 ± 1.3 for mounting, and 1.8 ± 0.6 to 0.2 ± 0.4 for ejaculation). That effect might due to the ability of the extract to stimulate the expression of eNOS and nNOS which results in nitric oxide production and subsequently maintains cyclic guanosine monophosphate levels in penile tissue. Moreover, this extract may also prevent penile tissue deterioration due to diabetes. Conclusions The polar extract of M. pruriens seed can be used for ED therapy, especially in patients with metabolic diseases including diabetes. The action of the extract might be due to catechol and its derivatives and polyphenols.