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In this study, we have reported a cationic amphiphilic peptide-based hydrogelator with a disulfide backbone and tryptophan side-chain residues that exhibited potent antimicrobial efficacy against several multidrug resistant bacterial and fungal strains. This C-terminally lipidated peptide was self-assembled into a thixotropic hydrogel in Tris-HCl buffer at physiological pH of 7.46. Subsequently, the morphology of the hydrogelators was well characterized by atomic force microscopy, field-emission gun transmission electron microscopy, and field-emission scanning electron microscopy, and it was apparent that the gelators formed a nanofibrous network. Bioactivity evaluation demonstrated that the peptide gelator solution showed broad-spectrum antibacterial activity with rapid bactericidal action and sustained bacteriostatic behavior against a wide range of Gram-positive and Gram-negative bacterial strains. In addition, pronounced antifungal activity was observed across multiple pathogenic fungal species. Detailed mechanistic study revealed a multimodal antimicrobial action involving membrane permeation, trans-membrane depolarization, and reactive oxygen species generation, which ultimately led to microbial cell flocculation. Interestingly, this injectable hydrogel also displayed strong antibiofilm performance against bacterial strains like methicillin-resistant Staphylococcus aureus and Klebsiella pneumoniae and also against fungal pathogens such as Aspergillus niger and fluconazole-resistant Candida albicans. Both extracellular matrix degradation and subsequent planktonic microbial decay effectively inhibited biofilm growth and disrupted mature biofilm microcolonies. Notably, the peptide hydrogel showed nonsignificant cytotoxicity toward normal eukaryotic cells such as HEK-293 (human embryonic kidney) and thus can be promoted as efficient biocompatible soft-material. Collectively, this study focuses on antimicrobial and antibiofilm properties of an injectable lipopeptide hydrogel, highlighting its potential as a promising biomaterial for anti-infective surface-coating and infection-resistant biomedical applications.<\/jats:p>","DOI":"10.1021\/acsabm.6c01362","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T08:01:52Z","timestamp":1789545712000},"page":"9186-9200","source":"Crossref","is-referenced-by-count":0,"title":["Cystine Containing Cationic Amphiphilic Peptide-Based Injectable, Noncytotoxic, Proteolytically Stable Hydrogel with Anti-infective Surface-Coating Activity against Multidrug-Resistant Bacterial and Fungal Biofilms"],"prefix":"10.1021","volume":"9","author":[{"given":"Supratim","family":"Bose","sequence":"first","affiliation":[{"name":"Indian Association for the Cultivation of Science , , 2A & Raja S. C. 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