Candida albicans is a major cause of fungal diseases in humans and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small scale screening of a 199 natural plant compounds (NPs) library. In vitro susceptibility profiling experiments identified 33 NPs with activity against C. albicans (MIC50 ≤ 32 µg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from Solanum lycopersicum (tomato) and exhibited high fungistatic activity against Candida species (MIC50 ≤ 1 µg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of C. albicans tomatidine-treated cells revealed a major alteration (upregulation) of ergosterol genes suggesting that the ergosterol pathway was targeted by this NP. Consistent with this transcriptional response, sterol content analysis of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in Saccharomyces cerevisiae coupled with whole genome sequencing identified 2 non-synonymous mutations in ERG6 (amino acids: D249G and G132D) responsible for tomatidine resistance. Our results therefore identified unambiguously Erg6, a sterol C-24 methyltransferase absent in mammals, as the main direct target of tomatidine. We tested the in vivo efficacy of tomatidine in a mouse model of C. albicans systemic infection. Treatment with a nano-crystal pharmacological formulation successfully decreased the fungal burden in infected kidneys as compared to placebo and thus confirmed the potential of tomatidine as a therapeutic agent