Fungal infections have increased in the past decades in developed countries. Because the therapeutic arsenal is limited and the occurrence of antifungal resistance is not likely to decrease, it is necessary to identify novel effective agents. The present work aimed to identify promising antifungal NPs against planktonic fungi cells and biofilms through a comprehensive approach. The strategy included chemical and biological profiling of natural extracts, targeted isolation of bioactive compounds and in-depth investigation of their bioactivity profiles. This led to the identification of 45 compounds from 7 well-selected crude plant extracts. A broad activity spectrum on various human and phyto-pathogenic fungal strains was recorded and their toxicity was assessed. In vivo activity on the Galleria mellonella model was carried out. Preliminary investigation of the action mechanisms was undertaken by evaluating cytological effects in treated strains. The isolated compounds and active compounds from the literature were used for ligand-based virtual screening.