In addition to widespread use in reducing the symptoms of colds and flu, Echinacea is traditionally employed to treat fungal and bacterial infections. However, to date the mechanism of antimicrobial activity of Echinacea extracts remains unclear. We utilized a set of ∼4,600 viable gene deletion mutants of Saccharomyces cerevisiae to identify mutations that increase sensitivity to Echinacea. Thus, a set of chemical-genetic profiles for 16 different Echinacea treatments was generated, from which a consensus set of 23 Echinacea-sensitive mutants was identified. Of the 23 mutants, only 16 have a reported function. Ten of these 16 are involved in cell wall integrity/structure suggesting that a target for Echinacea is the fungal cell wall. Follow-up analyses revealed an increase in sonication-associated cell death in the yeasts S. cerevisiae and Cryptococcus neoformans after Echinacea extract treatments. Furthermore, fluorescence microscopy showed that Echinacea-treated S. cerevisiae was significantly more prone to cell wall damage than non-treated cells. This study further demonstrates the potential of gene deletion arrays to understand natural product antifungal mode of action and provides compelling evidence that the fungal cell wall is a target of Echinacea extracts and may thus explain the utility of this phytomedicine in treating mycoses.