The fungus Saccharomyces cerevisiae (i.e., baker's yeast) has been a seminal model for studies of lysosomal trafficking, biogenesis, and function. Several yeast mutants defective in such vacuole events have also been shown to be hypersensitive to low levels of hygromycin B, an aminoglycoside antibiotic. We hypothesized that hypersensitivity to hygromycin B may be linked to vacuole defects and performed a genomic screen for growth hypersensitivity to hygromycin B (hhy mutants) using a haploid deletion strain library of non-essential genes. Fourteen deletion strains showed the phenotype. These hhy mutants were characterized with respect to integrity of the vesicular trafficking pathway, and to temperature sensitive growth. The results confirm defects in vacuole trafficking or function in the hhy mutants. Three of the genes were not previously associated with vesicular and/or vacuolar events: two cell cycle related genes PAF1 and TPD3, and a previously uncharacterized gene ENV6.