Urinary tract infection mainly occurs by microorganism when they overcome the human immune system. These microbes have caused the high rate of morbidity and motility effect all over the world to every age and gender. Lower urinary tract infection is more common among females than males. A prospective observational study was conducted in Pathology Department at Lady Reading Hospital Medical Teaching Institution, Peshawar, Pakistan in a 4th quarter (October, November, December) of the year 2017. Mid-stream urine samples were collected from 1357 patients from all hospital-admitted and outdoor patients. They were cultured on CLED agar, after isolation, uropathogens was identified through biochemical tests like oxidase, catalase and TSI tests. Only 205 samples were reported positive, with the prevalence of 15.10%. Females (51.70%) were more prone to UTI then males (48.29%). E.coli was most frequently isolated uropathogen with frequency of 90.24%. Other isolates were Pseudomonas aeruginosa (4.8%), Enterococcus faecium (2.92%), Enterobacter species (1.46%) and Proteus mirabilis (0.49%). Thirteen antibiotics were used to test the susceptibility of isolated microbes through Kirby Bauer Disc Diffusion method. Carbapenem drug like Imipenem (89.7%) was most effective against gram-negative microbes belonging to Enterobacteriaceae family. Other antibiotics like, Piperacillin + Tazobactam (84.86%), Cefoperazone /Sulbactam (84.3%), Amikacin (84.3%), Fosfomycin (83.7%) and Nitrofurantoin (77.2%) also showed sensitivity. Fluoroquinolones Drugs like Ciprofloxacin (82.1%) and Levofloxacin (81.6%) showed high resistivity followed by Co-trimoxazole (80.54%) and Cephalosporine drugs. For Gram positive Enterococcus spp, Glycopeptide antibiotics and protein synthesis inhibitor drugs had shown 100% sensitivity, but carbapenem and fluoroquinolones were highly resistant.