A series of bisquaternary ammonium salts with a 12-carbon spacer between the positive charges were synthesized, and their antifungal activity has been investigated. Compounds with butyl, pentyl, and isopentyl headgroups were the most potent antifungal agents with MICs in the range of 2.2-5.5 microM against both Cryptococcus neoformans and Candida albicans. The antifungal activity of these compounds correlated with their inhibition of cryptococcal phospholipase B1 (PLB1), a newly identified virulence factor. This indicates that the mode of action of these compounds may be inhibition of the fungal PLB1 enzyme, further validating this enzyme as a target for the development of novel antifungal therapies.