In case of Escherichia coli and Klebsiella pneumoniae infection, the increased prominence of multidrug-resistance strains has become the greatest challenge in the urinary tract disease treatment. Therefore, the 16S rRNA sequencing of multidrug-resistant strains was performed, in addition to those of plasmids and genes responsible for multidrug resistance. These strains showed containing responsible genes Sulfonamides sul1, Tetracycline Tet(A), Tetracycline Tet(B), chloramphenicol catA1, beta-lactams blaSHV, and cmlA. Also, the strains demonstrated resistance to at least 10 types of antibiotics or more due to carrying various plasmids. For increasing the level of public health in daily life and treatment of multidrug-resistant bacteria, the nanomedicine was employed. Consequently, ZnO nanoparticles (ZnONPs-E) were synthesized by employing supernatant of Escherichia hermannii strain isolated from raw milk source. The E. hermannii strain produces high concentration of ZnONPs-E compared to other strains so we used it in this study. This ZnONPs-E has a minimal inhibitory concentration (MIC) ranged from the concentration 10 mu g/ml to 40 mu g/ml against E. coli and K. pneumoniae, respectively. The antimicrobial efficiency of ZnONPs-E was 40 mu g/ml and it was superior to the reported values in literature. Moreover, SEM results evident for distorted membrane morphology, blebbing of membrane, cell elongation, and leakage of cellular contents due to ZnONPs-E activity against tested bacteria. These results indicated that the ZnONPs-E exhibited interesting antimicrobial activity against pathogenic extended-spectrum beta-lactamases (ESBLs) strains. The present study revealed that the active components entered in biosynthesis of ZnONPs-E pave the way to lead its effective nano-medical and drug delivery applications.