Petroleum ether and dichloromethane extracts of fruits, aerial parts and exudate of Zuccagnia punctata Cav. (Fabaceae) showed moderate antifungal activities against the yeasts C. albicans, S. cerevisiae and C. neoformans (MICs: 62.5 - 250 μg/mL) and very strong antifungal activities against the dermatophytes M. gypseum, T. rubrum and T. mentagrophytes (MICs: 8 - 16 μg/mL) thus supporting the ethnopharmacological use of this plant. Antifungal activity-directed fractionation of active extracts by using bioautography led to the isolation of 2′,4′-dihydroxy-3′-methoxychalcone (1) and 2′,4′-dihydroxychalcone (2) as the compounds responsible for the antifungal activity. Second-order studies included MIC₈₀, MIC₅₀ and MFC of both chalcones in an extended panel of clinical isolates of the most sensitive fungi, and also comprised a series of targeted assays. They showed that the most active chalcone 2 is fungicidal rather than fungistatic, does not disrupt the fungal membranes up to 4 × MFC and does not act by inhibiting the fungal cell wall. So, 2′,4′-dihydroxychalcone would act by a different mechanism of action than the antifungal drugs in current clinical use, such as amphotericin B, azoles or echinocandins, and thus appears to be very promising as a novel antifungal agent. ADCM: aerial part dichloromethane extract AMeOH: aerial part methanol extract APE: aerial part petrol extract CC: column chromatography DMSO: dimethyl sulfoxide EDCM: exudate dichloromethane extract FDCM: fruit dichloromethane extract FMeOH: fruit methanol extract FPE: fruit petrol extract MFC: minimum fungicidal concentration MIC: minimum inhibitory concentration MOPS: 3-(N-morpholino)propanesulfonic acid RDCM: root dichloromethane extract RMeOH: root methanol extract RPE: root petrol extract RPMI-1640: Roswell Park Memorial Institute culture medium SDA: Sabouraud-dextrose agar