Multidrug antibiotic resistance is a major threat to global health today. Neisseria gonorrhoeae (Ngo), which causes the STI gonorrhoea, is particularly notorious, having developed resistance to nearly all front-line antibiotics. This is fuelled by the bacterium’s natural competence and remarkable ability to acquire resistance through DNA uptake and horizontal gene transfer, highlighting the urgent need for alternative therapeutics beyond conventional targets. To address this, our research investigates the understudied glyoxalase pathway which detoxifies the highly cytotoxic metabolite methylglyoxal (MG), which is thought to be upregulated in macrophages and neutrophils during the host immune response. The glyoxalase pathway typically comprises of two enzymes, glyoxalase I or GloA and glyoxalase II or GloB, which detoxify MG to D-lactate in a two-step reaction and recycle glutathione in the process. Although this pathway has been studied in a few bacteria, it has not yet been characterized in Ngo and its role in pathogen survival and infection remains elusive. Interestingly, some bacteria have also been shown to possess more than one glyoxalase I or glyoxalase II enzyme, which may have additive effects in MGO detoxification. Here, we describe a putative GloB isoenzyme, Ngo_MBL, that may contribute to MG detoxification in Ngo. This work shifts from conventional approaches of studying essential bacterial processes or known resistance mechanisms to harnessing metabolic stress response pathways that may contribute to survival during host infection under conditions of oxidative stress and immune pressure. We use biochemical and genetic approaches characterize Ngo_MBL function and assess the significance of the glyoxalase system in Ngo. More specifically, we aim to determine how disrupting this pathway impacts growth, MG detoxification, oxidative stress tolerance and infection phenotypes in Ngo. By doing so, we can identify metabolic choke points that promote survival in the host environment and advance our understanding of the physiology of bacterial pathogens during infection