INTRODUCTION: The emergence of -resistant strains represents one of the most urgent global threats. In this regard, C7-3 peptide is one of the anti-virulence therapies that has demonstrated promising anti-gonococcal activity. Accordingly, this research aimed to formulate C7-3 peptide and its derivatives in chitosan nanoparticles. METHODS: The peptide loaded chitosan nanoparticles were prepared using ion gelation method, and their physicochemical characteristics were investigated. The anti-gonococcal and antibiofilm activity of prepared NPs was assessed, and their cytotoxicity in human ovarian cells was evaluated. RESULTS: All prepared NPs were optimized for the smallest particle size of 136.9 to 168.3 nm. The EE% of C7-3, C7-3m1, and C7-3m2 CNPs reached 90.2, 92.5, and 91.8%, respectively. An in vitro release study demonstrated a continuous sustained-release pattern of C7-3 peptide from NPs. The SDS-PAGE assay confirmed the integrity of C7-3 peptide after the fabrication process. When comparing each peptide alone, the generated NPs demonstrated higher anti-gonococcal and anti-biofilm effectiveness against standard and resistant bacterial strains under anaerobic conditions. The cytotoxicity experiments revealed the cytocompatibility of NPs in HeLa cell lines. Given the advantages of enhanced anti-gonococcal activity of the C7-3 peptide and its derivatives when loaded with CNPs, as well as the antimicrobial properties of chitosan NPs, the reported NPs have great potential in the treatment of gonococcal infection.