Campafungin A is a polyketide that was recognized in the  Candida albicans  fitness test due to its antiproliferative and antihyphal activity. Its mode of action was hypothesized to involve inhibition of a cAMP-dependent PKA pathway. The originally proposed structure appeared to require a polyketide assembled in a somewhat unusual fashion. However, structural characterization data were never formally published. This background stimulated a reinvestigation in which campafungin A and three closely related minor constituents were purified from fermentations of a strain of the ascomycete fungus  Plenodomus enteroleucus . Labeling studies, along with extensive NMR analysis, enabled assignment of a revised structure consistent with conventional polyketide synthetic machinery. The structure elucidation of campafungin A and new analogues encountered in this study, designated here as campafungins B, C, and D, is presented, along with a proposed biosynthetic route. The antimicrobial spectrum was expanded to methicillin-resistant  Staphylococcus aureus ,  Candida tropicalis ,  Candida glabrata ,  Cryptococcus neoformans ,  Aspergillus fumigatus , and  Schizosaccharomyces pombe , with MICs ranging as low as 4-8 μg mL -1  in  C. neoformans . Mode-of-action studies employing libraries of  C. neoformans  mutants indicated that multiple pathways were affected, but mutants in PKA/cAMP pathways were unaffected, indicating that the mode of action was distinct from that observed in  C. albicans. 