The bactericidal effect of HM-242, a novel antimicrobial agent, against Pseudomonas aeruginosa was investigated by using Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) values and the time-kill study. Furthermore, we also morphologically investigated its effect against P. aeruginosa by use of transmission electron microscopy (TEM) in comparison with that of chlorhexidine digluconate (CHG). The bactericidal activity of HM-242 after 1 min incubation evaluated by MBC was 25 microg/mL, while the MBC of CHG was 50 microg/mL. In the time-kill study, the killing activity of HM-242 with 25 microg/mL incubation was stronger than that of CHG with 50 microg/mL incubation. MIC values of HM-242 and CHG against P. aeruginosa were 25 microg/mL and 12.5 microg/mL, respectively. We also observed via TEM the morphological changes in the test bacteria after being treated with each drug at 1/2MBC, 1MBC, 2MBC and 4MBC after 1 min or 5 min incubation. Under treatment with the same concentration of the test drugs, the cell damage with HM-242 treatment was greater than that with CHG. The appearance of empty cells was recognized at the concentrations greater than 50 microg/mL (2MBC) of HM-242 and 200 microg/mL of CHG (4MBC) after 1 min exposure, although no cell damage was evident below these concentrations. The cell-damaging effect against the test strain was dependent on the drug concentration and incubation time. The release of cell components and bleb formation were also recognized. These results suggest that HM-242 has more potent bactericidal activity in low concentrations under shorter time treatments than CHG. Both HM-242 and CHG act on the cell membrane and cell wall of P. aeruginosa and can destroy the cell integrity. We finally emphasize that HM-242 as well as CHG might be a suitable disinfectant for use in the medical field.