Objectives The aims of this study were as follows: to determine the incidence of multidrug-resistant Enterobacteriaceae isolated from the urine of patients admitted in different departments at Menoufia University Hospitals; to detect the presence of extended spectrum β-lactamases (ESβLs), AmpC β-lactamases, and carbapenemases among the isolated pathogens using phenotypic methods; and to investigate the presence of bla KPC and bla NDM genes using real-time PCR. Background Urinary tract infections are the most common type of nosocomial infections. ESβLs and AmpC β-lactamases are clinically significant because they may confer resistance to multiple classes of antibiotics. Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams. Materials and methods This study included 260 urine samples collected from 260 patients. ESβL production was detected on the basis of resistance to third-generation cephalosporins and aztreonam and then confirmed using the combined disk test. AmpC production was detected using the cefoxitin disk test and confirmed using the AmpC disk test. Carbapenemases were detected on the basis of resistance to imipenem, meropenem, and ertapenem, and then confirmed using the modified Hodge test, the phenylboronic acid combined disk test, and the imipenem/EDTA combined disk test. Carbapenemase gene (bla KPC and bla NDM) detection was performed using real-time PCR. Results Carbapenem resistance was 42.5% to ertapenem, 40.8% to meropenem, and 45% to imipenem. Risk factors associated with Hospital aquired infection were hospital stay of 14 days or more, exposure to invasive procedures, and comorbid conditions. ESβL production occurred in 55.8% and AmpC occurred in 30.8% of Enterobacteriaceae spp. Carbapenemase production was detected in 87%, Klebsiella pneumoniae carbapenemase (KPC) was detected in 37%, metallo-β-lactamase was detected in 54%, and bla KPCgene was detected in 24% of imipenem-resistant Enterobacteriaceae spp. However, bla NDM gene was not detected in all tested isolates. Conclusion Surveys of the prevalence, antibacterial susceptibility patterns, and identification of resistance patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients.