Introduction: Urinary tract infection (UTI) is one of the most common bacterial infections encountered by clinician. Therefore, accurate identification of bacterial isolates and their antibiotic susceptibility is essential. 

Objective: To isolate the etiological bacterial pathogens causing UTI and to determine their antibiotic sensitivity in patients attending the Emergency and Al-shifa Maternity Hospitals in Misurata City-Libya. 

Patients and Methods: 250 urine specimens were collected from patients during the period from January, 1st to February, 15th 2016. The specimens were cultivated and identified according standard methods. 

Results: Only 25.2% of specimens showed a significant growth whereas 74.8% of specimens were either no growth or insignificant. The sum of isolated gram negative bacilli was 66.66%, while other bacteria represented (33.33%). Escherichia coli (E. Coli) was the most gram negative bacilli isolated (50%), and prevalence of gram negative bacilli was the highest among females 78.57%. Most of the pathogens were sensitive to Aminoglycosides: Amikacin and Gentamicin, but with lower sensitivity to Cephalosporins and high resistance to Ampicillin and to lesser degree toward other antibiotics.

Conclusion: Enterobacteriacea was the main cause of urinary tract infections, which recall intensification of education and cultivation programs, general & personal hygiene procedures. As drug resistance among microorganisms pathogens is an evolving process, routine surveillance to provide physicians knowledge on the updated and most effective treatment of UTIs.