Introduction and Hypothesis: Prior studies suggest that acute cystitis diagnosis using standard urine culture (SUC) may be suboptimal compared to multiplex polymerase chain reaction (mPCR). mPCR is hypothesized to deliver faster and more accurate results. Methods: This was a prospective, paired, within-subject study of female patients ≥ 60 years presenting with symptoms of acute cystitis. For all patients, urine samples were collected and SUC and mPCR results using the Vikor Urine-ID™ platform were obtained. Initial treatment was based on the results of the first available test, either SUC or mPCR. The primary outcome was time from initial presentation to correct antibiotic therapy. Secondary outcomes included patient perception of symptoms, antibiotic regimen changes, and discordance between the test results. Results: A total of 87 patients were eligible for analysis. Overall, there were 36 positive SUC (41.4%) and 71 positive mPCR (81.6%) results. Discordant results were noted in 43.7% of patients, which lead to treatment changes in 33.3% of patients. The time to correct antibiotic therapy was not significantly different based on initial treatment choice test (mPCR 67.0 h vs SUC 78.2 h, p = 0.16). However, in the case of positive SUC, the time to correct antibiotic therapy was significantly shorter for mPCR (mPCR 64.8 h vs SUC 79.5 h, p = 0.02). Non-Escherichia coli species, specifically Enterococcus faecalis, were more likely to be identified on mPCR (43.7%) than on SUC (8.3%), p < 0.001. Conclusions: mPCR may be an effective method of identifying urinary pathogens in symptomatic patients, particularly those with non-Escherichia coli species.