The prevalence of male reproductive disorders and testicular cancer is steadily increasing. Because the exposure to chemicals disrupting natural hormone action has been associated with these diseases, it is important to identify endocrine disrupting chemicals (EDCs) and their targets of action. Here, a 3D-structural database that can be applied for virtual screening approaches to facilitate the identification of EDCs was constructed. The database was screened using pharmacophores of 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3), which catalyzes the last step of testosterone synthesis in testicular Leydig cells and plays an essential role during male sexual development. Among other chemicals, benzophenone (BP) UV-filters were predicted as potential 17β-HSD3 inhibitors. Biological analyses revealed (2,4-dihydroxyphenyl)-phenylmethanone (also known as benzophenone-1, BP-1) as an inhibitor of human 17β-HSD3 (IC 50 1.05 μM). BP-1 also efficiently blocked conversion of androstenedione to testosterone by mouse and rat 17β-HSD3 in whole-organ enzyme assays. Moreover, BP-1 antagonized the testosterone-dependent activation of androgen receptors (IC 50 5.7 μM), suggesting synergistic anti-androgenic effects of BP-1 by preventing testosterone formation and blocking receptor activation. In addition, analyses of several commonly used UV-filters on estrogen- and androgen-metabolizing 17β-HSD enzymes revealed 3-benzylidene camphor (3-BC) and 4-methylbenzylidene camphor (4-MBC) as low micromolar 17β-HSD2 inhibitors. In conclusion, screening of virtual chemical structure libraries can facilitate the identification of compounds interfering with hormone action. The potential disruption of 17β-HSD enzyme function by the UV-filters BP-1, 3-BC and 4-MBC requires further investigation and should be considered for safety assessment of these chemicals.