Urinary tract infections (UTIs) are among the most common acquired bacterial infections in humans. The current gold standard method for identification of uropathogens in clinical laboratories is cultivation. However, culture-based assays have substantial drawbacks, including long turnaround time and limited culturability of many potential pathogens. Nanopore sequencing technology can overcome these limitations and detect pathogens while also providing reliable predictions of drug susceptibility in clinical samples. Here, we optimized a metagenomic nanopore sequencing (mNPS) test for pathogen detection and identification in urine samples of 76 patients with acute uncomplicated UTIs. We first used twenty of these samples to show that library preparation by the PCR Barcoding Kit (PBK) led to the highest agreement of positive results with gold standard clinical culture tests, and enabled antibiotic resistance detection in downstream analyses. We then compared the detection results of mNPS with those of culture-based diagnostics and found that mNPS sensitivity and specificity of detection were 86.7% [95% confidence interval (CI), 73.5–94.1%] and 96.8% (95% CI, 82.4–99.9%), respectively, indicating that the mNPS method is a valid approach for rapid and specific detection of UTI pathogens. The mNPS results also performed well at predicting antibiotic susceptibility phenotypes. These results demonstrate that our workflow can accurately diagnose UTI-causative pathogens and enable successful prediction of drug-resistant phenotypes within 6 h of sample receipt. Rapid mNPS testing is thus a promising clinical diagnostic tool for infectious diseases, based on clinical urine samples from UTI patients, and shows considerable potential for application in other clinical infections.