The public health complexity globally the pathogenic Enterobacteriaceae are particularly Escherichia coli and Klebsiella pneumoniae, from the hospital samples isolates of E. coli and Klebsiella spp. 520 samples were obtained from patients in prakasam (DT), Coastal Andhra, Andhra Pradesh, India. Identified under clinical laboratory condition, for microbial culture Blood agar and MacConkey agar used and identified for selected pathogens, different biochemical test was tested against microbes. Results are in urine sample examined for this study screened (520), among them culture +ve (250), β-lactamase (33), E. coli (15), Klebsiella (18) and others (217), in blood samples was collected and analyzed (250) culture +ve are (25), β-lactamase (13), E. coli (6), Klebsiella (4) and others (15). Pleural fluids (10) are evaluated among them culture +ve (3), β-lactamase (2), E. coli didn’t observed, Klebsiella (2) and others (1) also obtained (Fig. 6). In synovial fluid samples (10) screened and evaluated among them culture +ve (3), β-lactamase (3), E. coli didn’t observed, Klebsiella (3) and others also didn’t find out. In antibiotic sensitivity test, observed that sensitive to Piperacillin tazobactum and Sensitive to imepenam. Concluded that the urine samples collected and examined in different methods β-lactamase, E. coli didn’t observed, Klebsiella identified and characterized in different confirmatory analysis, use of these antibiotics is compulsory to decrease the extend of these challenging strains.