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ABSTRACT Nitroxoline (NTX) is an antibiotic approved for the treatment of uncomplicated urinary tract infections (UTIs) caused by Enterobacteriaceae like Escherichia coli, and it has been on the market for more than 50 years. Despite being in use longer than several other clinically relevant antibiotics, the resistance of clinical isolates against NTX has not evolved significantly. To better understand this observation, we performed a standardized in vitro evaluation of NTX in comparison to other UTI drugs with a focus on resistance development and especially their consequences. We have managed to generate low-level resistant mutants in E. coli and Klebsiella pneumoniae using a long-term exposure setup, and identified mutations in different efflux-related genes and in other pleiotropic regulators as well as the sensor histidine kinase envZ. Subsequently, mutants were characterized by metabolic and proteomic analyses confirming major effects of NTX resistance on both metabolism and differential protein expression of E. coli, which ultimately also translates into reduced in vitro motility and in vivo virulence of mutant strains as shown in a zebrafish larvae infection model. IMPORTANCE Antimicrobial resistance (AMR) poses a global threat and requires the exploration of underestimated treatment options. Nitroxoline, an effective broad-spectrum antibiotic, does not suffer from high resistance rates in the clinics but surprisingly, it is not heavily used yet. Our findings provide compelling evidence that Nitroxoline resistance renders bacteria unable to cause an infection in vivo, thereby reinvigorating the potential of Nitroxoline in combating AMR.