Aspirochlorine, a compound belonging to the gliotoxin family of compounds, exhibits antifungal and antibacterial activity but its mechanism of action remains unknown. In this study we show that aspirochlorine inhibits the pathogenic fungus Candida albicans by acting on fungal protein synthesis. The compound selectively inhibits cell-free protein synthesis when using a C. albicans system, but does not inhibit this synthesis in vitro when tested with bacterial and mammalian systems. Moreover, in intact C. albicans cells, aspirochlorine inhibits protein synthesis but does not inhibit chitin, DNA or glucan synthesis though at high concentrations some inhibition of RNA synthesis is observed. By contrast, in intact Bacillus subtilis cells, aspirochlorine did not inhibit protein, DNA, or cell wall synthesis though it significantly inhibited RNA synthesis. Furthermore, using heterologous systems (mammalian ribosomes and C. albicans cytosolic factors) the data suggest that the inhibitory action of aspirochlorine is not exerted through a direct interaction with C. albicans EF-1 or EF-2.