Two sets of novel multiheaded cationic amphiphiles bearing one, two, and three trimethylammonium headgroups (T1, T2, and T3) and pyridinium headgroups (P1, P2, and P3), have been synthesized and tested for antimicrobial activities against both Gram-positive and Gram-negative bacteria. The multicationic headgroups in these amphiphiles were attached covalently via scissile ester-type linkages. The results were compared with those for known surface-active, nonhydrolyzable compounds cetyltrimethylammonium bromide (CTAB) and cetylpyridinium bromide (CPB). The killing effects of the new single-headed amphiphiles (T1 and P1) were lower than those of CTAB and CPB, but with an increase in the number of headgroups in the amphiphiles, the killing effects increased for both sets of compounds. It was found that amphiphiles with triple headgroups (T3 and P3) were most active among all the amphiphiles, whereas amphiphile P1 had a very poor killing effect on both types of bacteria. The multiheaded pyridinium amphiphiles were more active compared to their trimethylammonium counterparts. The time needed to kill bacteria with multiheaded amphiphiles was significantly less than that of single-headed amphiphiles. Owing to the presence of a cleavable ester moiety, these new amphiphiles are hydrolyzed spontaneously at physiological conditions. This property enables them to be readily metabolized and therefore have the potential to be superior disinfectants and antiseptics for food and body surfaces.