The global spread of multidrug-resistant pathogenic fungi presents a serious threat to human health, necessitating the discovery of antifungals with unique modes of action¹. However, conventional activity-based screening for previously undescribed antibiotics has been hampered by the high-frequency rediscovery of known compounds and the lack of new antifungal targets². Here we report the discovery of a polyene antifungal antibiotic, mandimycin, using a phylogeny-guided natural-product discovery platform. Mandimycin is biosynthesized by the mand gene cluster, has evolved in a distinct manner from known polyene macrolide antibiotics and is modified with three deoxy sugars. It has demonstrated potent and broad-spectrum fungicidal activity against a wide range of multidrug-resistant fungal pathogens in both in vitro and in vivo settings. In contrast to known polyene macrolide antibiotics that target ergosterol, mandimycin has a unique mode of action that involves targeting various phospholipids in fungal cell membranes, resulting in the release of essential ions from fungal cells. This unique ability to bind multiple targets gives it robust fungicidal activity as well as the capability to evade resistance. The identification of mandimycin using the phylogeny-guided natural-product discovery strategy represents an important advancement in uncovering antimicrobial compounds with distinct modes of action, which could be developed to combat multidrug-resistant fungal pathogens.