Saccharomyces cerevisiae is a proven model to investigate the effects of small molecules and drugs on fungal and eukaryotic cells. In this study, the mode of action of an antifungal metabolite, 2,4-diacetylphloroglucinol (DAPG), was determined. Applying a combination of genetic and physiological approaches, it was established that this bacterial metabolite acts as a proton ionophore and dissipates the proton gradient across the mitochondrial membrane. The uncoupling of respiration and ATP synthesis ultimately leads to growth inhibition and is the primary toxic effect of DAPG. A genome-wide screen identified 154 DAPG-tolerant mutants and showed that there are many alterations in cellular metabolism that can confer at least some degree of tolerance to this uncoupler. One mutant, ydc1, was studied in some more detail as it displayed increased tolerance to both DAPG and the uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) and appears to be unconnected to other tolerant mutant strains. Deleting YDC1 alters sphingolipid homoeostasis in the cell, and we suggest here that this may be linked to reduced drug sensitivity. Sphingolipids and their derivatives are important eukaryotic signal molecules, and the observation that altering homoeostasis may affect yeast response to metabolic uncoupling agents raises some intriguing questions for future studies.