Aims: This study aims to investigate the extended-spectrum beta-lactamases (ESBLs)-producing Enterobacteriaceae in the human urine samples and to understand the potential influence of the contaminated water the urine donors use. Study Design and Methodology: One hundred urine samples from females and the same number from males were collected from donors of different age groups and analysed. One hundred urine samples from females and the same number from males were collected from donors of different age groups. The enterobacterial isolation was done on CLED agar medium. Their identification was carried out first by the enzymatic technic, then using MALDI–TOF MS method. ESBLs-producing enterobacteria were identified by searching the synergy between Clavulanic acid and 3rd generation Cephalosporins. The antimicrobial susceptibility tests were carried out using the disk diffusion method. Results: In male urine samples, the identified ESBLs-producing strains belong to E. coli, E. agglomerans and K. pneumoniae. They were in 11 samples: 3 from donors aged 20 years or less, 3 also from those aged 21 to 40 years as well as in those aged 41 to 60 years, and 2 in samples from donors aged over than 60 years. In female urine samples, the identified ESBLs-producing strains belong to Escherichia coli, Enterobacter agglomerans, Yersinia frederiksenii, Salmonella typhi and Klebsiella pneumoniae. They were in 27 samples: 3 from donors aged 20 years or less, 9 from those aged 21 to 40 years, 12 from those aged 41 to 60 years, and 3 from donors aged over than 60 years. All the strains were sensitive to Imipenem and resistant to Cefotaxim, Ceftazidin, Amoxicillin+Clavulanic acid and Nitrofurantoin. The Multi-Antibiotic Resistance (MAR) index varied from 0.416 (E. coli strains, youngest male donors) to 0.916 (E. coli strains, old female donors). Most of strains react differently from one antibiotic to another (P<0.05), with the exception of E. coli strains from most samples. Most of the identified ESBLs-producing species have been reported in surface and groundwater the population use. These water resources could play a role in the human urinary tract infections. Conclusion: There is a serious health problem with great MAR indices for all bacterial species identified. Treating water before use could reduce the viability and spreading of ESBLs-producing bacteria and genes into the population.