Previously, we have described a series of azole antifungals namely 3-(1,2,4-triazol-1-yl)flavanones (TFs) containing an N-(phenethyl)azole framework required for sterol 14α-demethylase (CYP51) inhibitory activity. Similar mechanism of action of azoles in fungi and protozoan parasites prompted us to investigate the potential effects of TFs against promastigote and amastigote forms of Leishmania major (L. major), as well as their toxicity against macrophages, apoptosis induction and in silico interactions with the target enzyme. All compounds showed more potent anti-parasitic activity against L. major in comparison with reference azole drug fluconazole and standard antileishmanial agent glucantime. Among the tested compounds, the 4-chloro derivative (TF-2) was found to be the most potent one, being about 13 times more potent than fluconazole against promastigotes. TF-2 decreased both mean infection rate of macrophages (MIR) and mean number of amastigotes per macrophages (MNAPM), significantly more than fluconazole (P < .001). Furthermore, the cytotoxicity assay against J774.A.1 macrophages revealed that this compound displays high selectivity against amastigotes over macrophages (SI = 30.21). The in silico study showed that TF-2 can properly accommodated in the active site of parasitic CYP51 and coordinated to the heme. The SAR analysis showed that the introduction of 4-chloro on 2-phenyl moiety results in the best profile of activity and selectivity. Accordingly, the compound TF-2 prototype can be considered as promising candidate for development of new antileishmanial agents. Display omitted