The gemini quaternary salt (gemini-QUAT) containing two pyridinium residues per molecule, 3,3'-(2,7-dioxaoctane)bis(1-decylpyridinium bromide) (3DOBP-4,10), exerted fungicidal activity against Saccharomyces cerevisiae accompanied by respiration inhibition and the cytoplasmic material leakage of ATP, magnesium, and potassium ions. We previously found that gemini-QUAT exerted bacterioclastic action against Escherichia coli by causing the rapid and abundant leakage of turbid materials from the cells. In addition, the first stage of the bacterioclastic action was the leakage of magnesium ions, outer membrane protein E, ATP, and lipopolysaccharides. Here, we investigated how the gemini-QUAT 3DOBP-4,10 exerts fungicidal action against S. cerevisiae. The results showed that that ≥ 0.4 μM 3DOBP-4,10 stopped respiration and that ≥ 3.0, 1.0 and 1.0 μM caused the leakage of cytoplasmic components ATP, magnesium and potassium ions, respectively. Scanning and transmission electron micrographs showed a preserved cell wall structure, whereas intracellular organelles were destroyed in cells incubated with 3DOBP-4,10. We postulated that 3DOBP-4,10 exerts its fungicidal action against S. cerevisiae not through cell wall destruction and protein leakage, but rather by penetrating the cell wall and disrupting the membranes of organelles.