Objectives This work aimed to evaluate the antifungal and cytotoxic activity of the EtOH extract and fractions of Banisteriopsis argyrophylla leaves, and to perform the identification of these bioactive metabolites. Methods The EtOAc fraction (EAF) obtained from the ethanolic extract of B. argyrophylla leaves showed better antifungal potential against Candida spp. In this fraction, ten flavonoids have been identified by UHPLC‐ESI‐MSn. Then, EAF was submitted to column chromatography to give four new fractions (A1–A4). The cytotoxicity was determined against Vero cells. Key findings The EAF showed better antifungal potential against Candida spp. with minimum inhibitory concentrations (MICs) between 31.25 and 93.75 μg/ml. The (–)‐catechin (fraction A1) showed a MIC of 2.83 μg/ml against Candida glabrata. Fractions A2, A3 and A4 were rich in quercetins and kaempferols and showed good inhibitory concentrations (5.86–46.87 μg/ml) against C. albicans, C. glabrata and C. tropicalis. Conclusions The EtOH extract, fractions and the isolated (–)‐catechin showed lower toxicity to Vero cells than cisplatin, used as a positive control. Thus, the leaves of B. argyrophylla are a promising source of antifungal agents.