The environmentally-friendly antibiotic phenazine-1-carboxylic acid (PCA), which is primarily produced by Pseudomonas sp., has been found to effectively protect against various fungal pathogens. However, the mechanism by which PCA inhibits or kills fungal pathogens remains unclear. In this study, we tested whether the budding yeast, S. cerevisiae, is suitable for exploring the PCA target on fungi.We found PCA inhibited or killed yeast in a dose- and pH-dependent manner. Our further experiments showed that PCA treatment significantly disrupted vesicle trafficking (displayed by Snc1 and CPY localizations) and autophagy, quite differently from hygromycin B treatment did. We endeavored to explore the PCA target on budding yeast through yeast mutant library screening and microarray analysis. When many yeast mutants were hyper sensitive to PCA treatment, only a small number of yeast mutants were slightly resistant to PCA treatment. Microarray analysis showed global changes in mRNA levels to PCA treated yeast, but even the genes with the top ratio changes in mRNA level did not show a close association for their mutations to PCA sensitivity. We conclude that PCA treatment had a broad effect on yeast metabolism, so that the yeast mutant library screening and microarray analysis are not enough to figure out the specific PCA target(s) in budding yeast to further test on other fungi. Other techniques should be sought for exploring PCA target on fungi.