Bactericidal activity of grapefruit seed extract (GSE)-based disinfectant, as a safe disinfectant, was measured against five bacteria by Korean Food & Drug Administration (KFDA) dilution-neutralization method. GSE-based disinfectant showed a 99.9999% bactericidal activity against Escherichia coli ATCC 10536, Salmonella typhi ATCC 29629, Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 11778, and Listeria monocytogenes ATCC 1911 at the concentration of 2.15% GSE. It showed better bactericidal activity against Gram-negative bacteria of E. coli ATCC 10536 and S. typhi ATCC 29629 at lower concentration of GSE (0.43%). Based on the results, it was suggested that a possible bactericidal mechanism of GSE active ingredients was due to the abrupt osmotic shift during the bactericidal activity test by KFDA method.