Due to the increasing incidence of antibiotic-resistant bacterial pathogens, the need for the development of alternative compounds is of utmost importance. Bacteriocins are considered a promising alternative to antibiotics. In this study, we determined the in vitro activities of 65 bacteriocins from the PARAGEN collection along with microbisporicin (NAI-107) against 14 WHO reference isolates of Neisseria gonorrhoeae, one isolate of Neisseria meningitidis, and four commensal Neisseria spp. The initial screening of the bacteriocins was carried out using a spot-on-lawn assay wherein 12 bacteriocins (12/66) showed a zone of inhibition against the WHO-P N. gonorrhoeae isolate. Four bacteriocins (4/12), Garvicin KS (GarKS) (ABC), lacticin (lcn) Q, lcn Z, and NAI-107, demonstrated higher activity, and minimum inhibitory concentrations (MICs) using agar dilutions were determined for 19 Neisseria spp. GarKS (ABC), lcn Q, and lcn Z had an MIC range of 4-16 µg/mL, whereas NAI-107 had an MIC range of <0.25 to 2 µg/mL. Using an in vivo Galleria mellonella model, we determined the efficacy of GarKS (ABC), lcn Z, and NAI-107. GarKS (ABC) and lcn Z displayed no in vivo toxicity but showed no in vivo activity against WHO-P N. gonorrhoeae. In contrast, NAI-107 was as effective as ceftriaxone and significantly increased the survival rate of WHO-P N. gonorrhoeae-infected G. mellonella larvae. In conclusion, NAI-107 exhibited potent in vitro and in vivo activities against N. gonorrhoeae. To our knowledge, this is the first study to demonstrate that NAI-107 possesses anti-gonococcal activity in vivo. IMPORTANCE We screened 66 bacteriocins to see if they exhibited anti-gonococcal activity. We found 12 bacteriocins with anti-gonococcal effects, and 4 bacteriocins showed higher anti-gonococcal activity. Three bacteriocins, lacticin Z, lacticin Q, and Garvicin KS (ABC), showed in vitro anti-gonococcal activity but no in vivo inhibitory effects against the Neisseria gonorrhoeae (WHO-P) isolate. On the other hand, NAI-107 showed in vivo anti-gonococcal activity. The findings suggest that NAI-107 is a promising alternative to treat gonorrhea infections.