The incidence of resistance to nalidixic acid has increased in the University General Hospital of Elche, Spain, and this paper describes the investigation of this phenomenon. This increase was mainly due to an increase of nalidixic-resistant Salmonella enterica serotype Enteritidis. Resistance to nalidixic acid is an indicator of decreased ciprofloxacin susceptibility (sensitivity 100%, specificity 96.7%). Strains that were resistant to nalidixic acid and exhibited decreased susceptibility to ciprofloxacin had a single mutation in QRDR of gyrA: Asp87-Asn, Asp87-Tyr or Ser83-Phe. The sensitivity of S. enterica strains to nalidixic acid should be tested and the breakpoint of ciprofloxacin established by MENSURA applied, instead of that of the NCCLS for these strains.