Infections by drug-resistant fungi are increasingly reported worldwide; however, only few novel antifungals are being developed. The old antimicrobial nitroxoline is currently repurposed for oral treatment of bacterial urinary tract infections (UTI). Previously, antifungal activity has been demonstrated and in contrast to many antifungals nitroxoline reaches high urinary concentrations. In this study, the activity of nitroxoline was assessed in vitro in a collection of yeasts from the German National Reference Centre for Invasive Fungal Infections. Susceptibility was determined by broth microdilution (BMD) and disk diffusion (DD). The collection comprised 45 Candida isolates originating from the urinary tract. MICs of amphotericin, anidulafungin and azoles were analyzed using EUCAST BMD. Among the collection isolates, resistance to antifungals was common, e.g., for fluconazole the MIC50/90 was 16/>64 mg/L; in contrast MIC50/90 of nitroxoline was 2/2 mg/L (MIC range 0.25-4 mg/L), which is at least two dilutions below the EUCAST breakpoint for uncomplicated UTI defined for E. coli (susceptible ≤ 16mg/L). Activity of nitroxoline was high irrespective of resistance to other agents. As BMD is labor-intensive, DD was investigated as an alternative method using three different agars. Nitroxoline disks produced large inhibition zones on all agars (≥19mm), but the correlation of MICs and zone diameters was low, with the highest correlation recorded for the CLSI recommended agar for antifungal DD (Pearson's r = -0,52). In conclusion, isolates of different Candida species are highly susceptible to nitroxoline, which could be a promising antimicrobial to treat candiduria caused by multidrug resistant yeasts.