Background: Extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae threaten infection treatment globally. This study aims to assess ESBLs-E prevalence and multidrug-resistant organisms (MDR) in clinical specimens from Tabuk, KSA.Methods: A cross-sectional research was carried out in March-May 2023. A collective of 90 Enterobacteriaceae isolates were identified from clinical specimens. The specimen was identified by standard methods. The Enterobacteriaceae member was screed for ESBL production by screening and confirmatory as per the Clinical and Laboratory Standards Institute (CLSI).Result:E. coliwas the most common isolate, followed byProteus mirabilis and Citrobacter sp,Klebsiella oxytoca,Klebsiella pneumonia,Proteus vulgarisandMorganella morganii. Among the sample, the majority of isolates were from urine (47.8%) followed by pus (25.6%) and the least from other body fluids (6.7%). TheE. colishowed the highest average antibiotic resistance (73.7%) among all the antibiotics used followed byP. mirabilis(70.4%),K. pneumoniae(70%),P. vulgaris(69.8%),M. morganiiandCitrobacter(69.4% both), andK. oxytoca(68.8%). There was a 41.2% average reduction in ESBL positivity from phenotypic to confirmatory test results. The highest reduction was observed amongM. morganii(66.7%) and the least was observed inE. coli(17.1%).Conclusion: Most of the ESBL-producing isolates were found mainly in blood and urine samples. The most frequent ESBL-producing Enterobacteriaceae wereK. pneumoniaeandE. coli. The best options for treating Enterobacteriaceae that produce ESBL are Amoxicillin, Amikacin, and Cefoxitin. ESBL-producing isotopes showed a high resistance rate to cefepime and cefotaxime compared to non-ESBL producers. It is of utmost importance to implement reliable infection control measures in healthcare institutions nationwide.