A simple in vitro agar disk diffusion assay has been employed to detect the presence of ergosterol-sensitive antifungal activity in extracts of marine invertebrates. A collection of 116 marine sponges, ascidians, and cnidarians was assayed to reveal 10 samples (8.3%) with significant activity against Candida albicans. The antifungal activities of three of these extracts were unaffected in the presence of increasing concentrations of ergosterol, while another three were significantly reduced by ergosterol. The activity of jaspamide [3], a potent antifungal from the sponge Jaspis sp., was also reduced by ergosterol concentrations as low as 10 ppm. This ergosterol sensitivity is paralleled by the well-known ergosterol dependence of polyene antifungals, such as amphotericin B [1], and suggests a common mode of activity. The assay may be useful in mechanism-selective screening for new antifungals and as a dereplication tool.