PURPOSE: The emergence of strains that are resistant to the most commonly used antibiotics represents a great concern for global public health. This challenges the effectiveness of clinical treatment regimens and demands the development of alternative antigonococcal agent. In this regard, chitosan nanoparticles (CNPs) are known to have antimicrobial activity against a wide range of pathogens. Thus, they have become a potential candidate for combatting this era of multi-drug resistance. This study aims to formulate CNPs, characterize their physicochemical properties, and examine their antimicrobial activity against gonococcus. MATERIALS AND METHODS: The ionic gelation method was used to prepare CNPs of different concentrations. Characterization for their particle size (PZ), polydispersity index (PDI), and zeta potential (ZP) was performed. The anti-microbial activity of CNPs was investigated against 13 WHO reference strains, using the broth dilution method. Cytotoxicity of CNPs and their effect on bacterial adhesion to HeLa cells were investigated. RESULTS: The average PZ and ZP of the prepared NPs were increased when the concentration of chitosan was increased from 1 to 5 mg/mL and found to be in the range of 193 nm ± 1.9 to 530 nm ± 13.3, and 14 mV ± 0.5 to 20 mV ± 1, respectively. Transmission electron microscopes (TEM) images revealed spherical NPs, and the NPs had a low PDI value of ≤0.27. The formed CNPs produced antibacterial activity against all tested strains, including those resistant to multiple antibiotics, with a minimum inhibitory concentration (MIC) of 0.16 to 0.31 mg/mL and a minimum bactericidal concentration (MBC) of 0.31 to 0.61 mg/mL. Of note, at all MIC and MBC, the CNPs had no significant cytotoxic effect on HeLa cells and reduced bacterial adhesion to these cells at MBC doses. CONCLUSION: The present work findings suggest the potential of the CNPs for the treatment of gonorrhoea.