Urinary tract infections are the leading cause of human infectious disease and prevailing rapidly. UTIs are the most common infections to humans, and large female populations get infected once in their lives. There are growing research evidence of juvenile cases of UTIs with a higher rate of deformities. India and many other developing nations reported a higher prevalence of UTI cases due to poor hygiene and sanitation. The present work was carried out in the City of Bhopal, Madhya Pradesh, India, during 2014–2015. The screening of uropathogens has revealed E. coli as key uropathogens followed by Klebsiella species. The gram staining, microscopy and biochemical characterization studies have shown present of E. coli (62%), klebsiella (14%), Pseudomonas (3%), Enterobacter (11%) and Proteus (1%) along with Staphylococcus species (3%). We report here E. coli had gained tremendous antibiotics resistance against Nitrofurantoin, Ciprofloxacin, and Co-Trimoxazole. The total MDR percentage for E. coli in the given population was noted 36.00% while XDR and PDR were reported at 11.00% and 2.0%. The other two uropathogens, Klebsiella and Enterobacter, were reported with significantly higher drug resistance in the present study. The percentage of drug resistance for Klebsiella (MDR (67.25%), XDR (24.30%), and PDR (2.36%)) while in the case of Enterobacter percentage of MDR (74.55%), XDR (11.24%) and PDR (7.58%) remain slightly higher. We also report that the Staphylococcus also succeeded in gaining resistance while Pseudomonas and Proteus showed negligible drug resistance. The molecular investigations have shown a high frequency of gyr-A, tet-A gene, and aac (6′)-Ib genes in uropathogens isolated in the present study. Other genes were also present but in lower frequencies. We have also found a high prevalence of β-lactamase gene in 1/4th population of uropathogens isolated from the present study.