Infections of the urinary tract are one of the most prevalent bacterial infections in developing countries. It is a challenge that is most often encountered in pediatrics. Uncontrolled use of antimicrobial drugs has led to the development of drug-resistant organisms. Hence, this study was aimed at isolating, characterizing, and identifying the pathogens associated with urinary tract infection (UTI) in children attending Tertiary Health Care Centre in Enugu metropolis and to determine their antimicrobial susceptibility profiles. Urine samples were collected from 260 patients with signs and symptoms suggestive of urinary tract infection (UTI). The children were seen in children out-patients, children emergency departments of the hospital, and pediatric ward. Their ages ranged from 0-17 years. The urine samples were analyzed in the laboratory using standard bacteriological methods. Antibiotic sensitivity of the isolates was determined by the Kirby-Bauer disc diffusion technique. Of the 260 urine samples, significant bacterial agents were recorded in 98(37.7%). Escherichia coli ranked highest with 30(32.6%) followed by Klebsiella spp. 17(18.5%) and the least was Enterococcus fecalis 1(1.1%). Gender distribution showed a higher prevalence in females (p <0.05). The highest positive result was obtained from children between 0-5years. Most of the isolates were sensitive to Imipenem, Nitrofurantoin, Ceftriaxone, and Ciprofloxacin but resistant to Augmentin and Amoxicillin. There is a need to regularly monitor the antimicrobial susceptibility profile of these etiological agents to keep track of the effectiveness of certain therapeutic agents.