It is predicted that failure to address the antibiotic-resistance crisis will result in an annual death rate of 10 million people by the year 2050. To grapple with the challenges of the impending crisis, there is an urgent need for novel anti-bacterial agents and rapid diagnostic tools. Here, we developed a novel monoclonal antibody – known as mAb-EspB-B7 – that targets EspB, a component within the bacterial type 3 secretion system (T3SS), which is mainly expressed in Gram-negative pathogens and is essential for bacterial infectivity. We found that mAb-EspB-B7 has high affinity and specificity towards recombinant and native EspB proteins, is stable over a range of pH levels, temperatures and salt concentrations, and retains its functionality in human serum. We identified the epitope for mAb-EspB-B7 and validated it by competitive ELISA. Since this epitope is conserved across several T3SS-harboring pathogens, mAb-EspB-B7 holds great potential for development as an anti-bacterial agent or as the active component in precise and rapid diagnostic tools.