Purpose: Urinary Tract Infections (UTIs) are among the most prevalent infections globally. Every year, approximately 150 million people are diagnosed with UTIs worldwide. The current state-of-the-art diagnostic methods are culture-based and have a turnaround time of 2–4 days for pathogen identification and susceptibility testing. Methods: This study first establishes an optical density culture-based method for spiking healthy urine samples with the six most prevalent uropathogens. Urine samples were spiked at clinically significant concentrations of 10³-10⁵ CFU/ml. Three DNA extraction kits (BioStic, PowerFood, and Blood and Tissue) were investigated based on the DNA yield, average processing time, elution volume, and the average cost incurred per extraction. After DNA extraction, the samples were sequenced using MinION and Flongle flow cells. Results: The Blood and Tissue kit outperformed the other kits based on the investigated parameters. Using nanopore sequencing, all the pathogens and corresponding genes were only identified at a spike concentration of 10⁵ CFU/ml, achieved after 10 min and 3 hours of sequencing, respectively. However, some pathogens and antibiotic-resistance genes (ARG) could be identified from spikes at 10³ colony formation units (CFU/mL). The overall turnaround time was five hours, from sample preparation to sequencing-based identification of pathogen ID and antimicrobial resistance genes. Conclusion: This study demonstrates excellent promise in reducing the time required for informed antibiotic administration from 48 to 72 h to five hours, thereby reducing the number of empirical doses and increasing the chance of saving lives.