The aim of this study was to compare the in vitro activity of ethanol, EDTA and levofloxacin (Levo), alone or in combination, on biofilms of Stenotrophomonas maltophilia recovered from patients with catheter-related bloodstream infections (CRBSIs) at a university hospital in Argentina. First, 24 and 48 h biofilms were formed in microtitre plates and challenged with 25 or 40 % ethanol for 1 h. Biofilms, of the 14 local isolates and from the reference strain K279a, were eradicated after both treatments as shown by plate counts and the regrowth technique. Second, 24 h biofilms of all isolates were established in silicone catheter segments and challenged with 25 or 40 % ethanol, Levo (2.5 mg ml ), 25 % ethanol-EDTA or Levo-EDTA for 1, 3 and 24 h. Viable counts of biofilms treated for 1 h with 25 or 40 % ethanol or 25 % ethanol-EDTA were under the limit of detection. Killing of biofilms by Levo or Levo-EDTA was gradual and it was only after 24 h of treatment that no differences could be seen between the effects of these catheter lock solutions (CLSs) and those of ethanol (P.0.05). Levo-EDTA, in combination, did not act synergistically against biofilms. After 24 h of exposure, EDTA did not eradicate biofilms but reduced biofilm survival rates to 1-5 %. The effect of the different CLSs on biomass reduction, estimated by crystal violet staining, was highly dependent on the isolate, and the most effective agents were 25 and 40 % ethanol. Our results suggest that when used as a CLS for short periods, ethanol at low concentrations, alone or in combination with a chelator, can decontaminate the line from S. maltophilia in cases of CRBSI and help, in conjunction with systemic antibiotics, in the retention of precious vascular catheters.