Wounds or tissue openings in the skin are susceptible to bacterial attack, which can deteriorate and slow down the healing process. In this regard, antimicrobial gels are valuable as they mitigate the infection spread and assist in the healing. Despite the success, commercially available release-active antimicrobial gels suffer from narrow-spectrum activity, resistance induction, reservoir exhaustion, and in some cases may be associated with toxicity. To circumvent these limitations, herein, we have developed new quaternary lipophilic chitosan derivatives (QuaChi) synthesized by modifying the primary alcohol of the sugar moieties without altering the free amino groups of glucosamines. Compared to protonated chitosan, the synthesized derivatives exhibited improved water solubility and enhanced antibacterial activity against multidrug-resistant Gram-positive and Gram-negative bacteria including clinical isolates. The enhanced antibacterial activity was evident from the bacterial membrane depolarization leading to rapid inactivation of similar to 10(5)-10(6) bacterial cells within 2 h. The applicability of the chitosan derivatives was further demonstrated by developing antibacterial hydrogels by cross-linking the free amino groups of QuaChi with biocompatible gelatin through amide linkages. The hydrogel showed similar to 5-7 log reduction of various multidrug-resistant bacteria including the stationary-phase cells within 6 h. Scanning electron microscopy revealed the loss of integrity of the bacterial structure when treated with the hydrogel, whereas mammalian cells (human embryonic kidney-293 (HEK-293)), when exposed to the hydrogel, appeared to be healthy with retained morphology. Collectively, these findings suggest that the developed hydrogel formulation can find potential applications to combat notorious drug-resistant bacterial infections in the healthcare settings.