Development of biomaterials, which are inherently antibacterial having broad-spectrum activity against both Gram-positive and Gram-negative bacteria with considerable biocompatibility, is of tremendous importance in biomedicinal chemistry. Microbial infections are still of great concern, often originated from indwelling medical devices typically in hospitalized patients. To this end, hydrogelating soft materials particularly from low-molecular-weight (LMW) gelators have generated significant interest in preparing and modifying biomedicinal implants. Herein, we have developed L-tryptophan based cationic amphiphilic hydrogelators with varying degree of hydrophobicity that exhibited remarkable bactericidal activity against wide range of Gram-positive (MIC = 0.1–75 µg/mL) and Gram-negative bacteria (MIC = 0.5–5 µg/mL). Antimicrobial efficacy of the amphiphiles was greatly influenced by their alkyl chain length. This bactericidal effect of cationic hydrogelators is quite comparable or in some cases markedly better than that of clinically available antibiotics. Most excitingly, they selectively attack the bacterial pathogens while remain biocompatible to the mammalian cells. Thus, we have developed LMW biocompatible, inherently antibacterial hydrogels having potential applications in biomedicines. Biotechnol. Bioeng. 2008;100: 756–764. © 2008 Wiley Periodicals, Inc.