Drug-resistant fungus infections were becoming more common worldwide, yet only a few new antifungals were being produced. The ancient antibacterial nitroxoline was used to treat bacterial urinary tract infections (UTI) by mouth. The study evaluated nitroxoline activity in vitro in a collection of yeasts from the German National Reference Centre for Invasive Fungal Infections. Broth microdilution (BMD) and disc diffusion (DD) assessed susceptibility. About 45 Candida isolates from the urinary tract were included in the collection. EUCAST BMD was used to determine the MICs of amphotericin, anidulafungin, and azoles. Resistance to antifungals was common among the collection isolates; for example, the MIC50/90 of fluconazole was 16/ more than 64 mg/L; however, the MIC50/90 of nitroxoline was 2/2 mg/L (MIC range 0.25–4 mg/L), which was at least two dilutions below the EUCAST breakpoint for uncomplicated UTI defined for E. coli (susceptible ≤16 mg/L). Regardless of resistance to other drugs, nitroxoline had a strong activity. Because BMD was time-consuming, three different agars were used to test DD as an alternative. On all agars, nitroxoline discs produced large inhibition zones (≥19mm). However, the correlation between MICs and zone diameters was modest, with the CLSI-recommended agar for antifungal DD having the strongest correlation (Pearson’s r = −0,52). Finally, nitroxoline was extremely responsive to isolates of many Candida species, suggesting it could be a potent antibiotic for treating candiduria caused by multidrug-resistant yeasts.