Abstract The aim of this study was to evaluate the possible in vitro effects of Anethum graveolens L. (dill) extract on the steroidogenesis in testicular tissue. Specifically, we focused to determine the dominant chemical components of the dill extract followed by a subsequent assessment of its in vitro effects on the production of cholesterol, dehydroepiandrosterone, androstenedione and testosterone by rat testicular fragments. Dill extract was subjected to high performance liquid chromatography (HPLC) which determined cyranosid, carvone, chlorogenic acid, p-hydroxybenzoic acid and salicylic acid among the predominant biomolecules. For the in vitro experiments, testicular fragments from 12 sexually mature Wistar rats were incubated in the absence (control) or presence of 10, 100 or 1000 μg/mL extract for 24 h. Cholesterol levels were quantified via photometry and the hormones were evaluated by Enzyme-Linked Immunosorbent Assay (ELISA). Our data showed that the release of cholesterol and androstenedione (but not dehydroepiandrosterone and testosterone) by the testicular fragments obtained from seminiferous tubules was significantly affected by the dill extract in a dose-dependent manner. Exposure to the extract led to a significant decrease of both cholesterol as well as androstenedione (P<0.01 in case of 10 μg/mL; P<0.001 with respect to 100 and 1000 ug/mL extract). Our results suggest that the bioactive molecules present in Anethum graveolens L. could have dose-dependent impact on the secretion of selected male reproductive hormones, playing a role in the regulation of testicular steroidogenesis. Nevertheless, the exact effect of individual biomolecules on the endocrine testicular function has yet to be investigated.