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An epidemiological survey and evaluation of the anti-bacterial growth effect of the aqueous leaf extract of the traditional medicinal plant Rhazya stricta were undertaken for extended-spectrum β-lactamase’s producing bacteria isolates collected from the King Abdulaziz Hospital and Oncology Center. Results showed high rates of extended-spectrum β-lactamase (ESBL) isolates (49.2%) compared to non-ESBL isolates (50.8%). The surgical intensive care unit was the hospital site showing the highest rate (15.1%) of infection, followed by the women's medical ward (11.2%). Urine samples were the highest source of infection (40%). Prevalence among Saudis was 66.67% as compared to non-Saudis (33.33%). Prevalence according to gender was 51.94% females and 48.06% males. Age groups 51-60 years, 21-30 years, and 61 years and over showed the highest rates (20.93%, 20.54% and 20.16%, respectively) compared with other age groups. E. coli resistance rates were 94.5% for ceftazidime, 98.18% for cefotaxime, 92.73% for cefepime and 93.75% for aztreonam. On the other hand, in cases of K. pneumoniae resistance, rates were 100% for ceftazidime, 100% for cefotaxime, 98.68% for cefepime and 97.29% for aztreonam. Genetic analysis showed 8 genotypes, 4 of them were for E. coli and 4 for K. pneumoniae. Analysis also showed genetic diversities between the ESBL and the non-ESBL types in their genomic DNA. There was no antimicrobial effect of the plant extract against all study isolates, although the antimicrobial growth had an effect upon some of the control strains. Full article