Neisseria gonorrhoeae, the etiological agent of gonorrhea, remains a significant global health threat, exacerbated by rising antibiotic resistance. For its survival, N. gonorrhoeae utilizes the TonB-dependent transporters to acquire metals from the host. Zinc acquisition is essential for the bacterium's survival, with the TonB-dependent transporter TdfH playing a pivotal role by binding to human calprotectin to extract and acquire zinc thereby circumventing host-imposed nutritional immunity. This study aimed to map the epitopes of TdfH recognized by monoclonal antibodies (mAbs) using an overlapping peptide-ELISA approach. Four mAbs—5H-1C2, 4H-3B11, 5H-1E10, and 5H-6B12—were used to probe synthesized overlapping TdfH peptides, identifying distinct immunodominant regions. mAbs 5H-1C2 and 4H-3B11, belonging to the IgG1 subclass, recognized overlapping epitopes on peptides 61, 62, and 63 with common amino acid sequence NLAPQYDITP. While mAbs 5H-1E10 and 5H-6B12 (IgG2a) both recognized peptide 178 with the amino acid sequence CNDDNTLCNGKYGGT. Notably, peptides 176 and 177 were missing due to peptide synthesis challenges. The conserved nature of these epitopes among disseminated and urogenital strains of N. gonorrhoea underscores these region as a viable target for immune-based interventions, with implications for both therapeutic monoclonal antibody development and vaccine design. This work advances the understanding of N. gonorrhoeae immune evasion strategies and provides a framework for developing novel therapeutic interventions, particularly in the context of rising antibiotic resistance.