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Nanoparticles (NPs) possessing antibacterial activity represent an effective way of overcoming bacterial resistance. In the present work, we report a novel formulation of a nanoantibiotic formed using Ampicillin/sulbactam (Ams) and a zinc oxide nanoparticle (ZnO NP). &#8216;ZnO NP&#8722;Ams&#8217; nanoantibiotic formulation is optimized using response surface methodology coupled genetic algorithm approach. The optimized formulation of nanoantibiotic (ZnO NP: 49.9 &#956;g/mL; Ams: 33.6 &#956;g/mL; incubation time: 27 h) demonstrated 15% enhanced activity compared to the unoptimized formulation against <i>K. pneumoniae</i>. The reactive oxygen species (ROS) generation was directly proportional to the interaction time of nanoantibiotic and <i>K. pneumoniae</i> after the initial lag phase of ~18 h as evident from 2'-7'-Dichlorodihydrofluorescein diacetate assay. A low minimum inhibitory concentration (6.25 &#956;g/mL) of nanoantibiotic formulation reveals that even a low concentration of nanoantibiotic can prove to be effective against <i>K. pneumoniae.</i> The importance of nanoantibiotic formulation is also evident by the fact that the 100 &#956;g/mL of Ams and 25 &#181;g of ZnO NP was required individually to inhibit the growth of <i>K. pneumonia</i>,<b> </b>whereas only 6.25 &#956;g/mL of optimized nanoantibiotic formulation (ZnO NP and Ams in the ratio of 49.9: 33.6 in &#956;g/mL and conjugation time of 27 h) was needed for the same.