Leaves of guava (Psidium guajava L.) have been used in Thai folk medicine without any supporting evidence as a traditional herbal remedy for hair loss. Androgenetic alopecia (AGA) is chronic hair loss caused by effects of androgens in those with a genetic predisposition, resulting in hair follicle miniaturization. Our objectives were to provide the mechanistic assessment of guava leaf extract on gene expressions related to the androgen pathway in well-known in vitro models, hair follicle dermal papilla cells (HFDPC), and human prostate cancer cells (DU-145), and to determine its bioactive constituents and antioxidant activities. LC-MS analysis demonstrated that the main components of the ethanolic extract of guava leaves are phenolic substances, specifically catechin, gallic acid, and quercetin, which contribute to its scavenging and metal chelating abilities. The guava leaf extract substantially downregulated SRD5A1, SRD5A2, and SRD5A3 genes in the DU-145 model, suggesting that the extract could minimize hair loss by inhibiting the synthesis of a potent androgen (dihydrotestosterone). SRD5A suppression by gallic acid and quercetin was verified. Our study reveals new perspectives on guava leaf extract's anti-androgen properties. This extract could be developed as alternative products or therapeutic adjuvants for the treatment of AGA and other androgen-related disorders.