Biofilms on catheters are responsible for catheter-related bloodstream infections (CRBSIs), which cause significant mortality and morbidity. Antimicrobial catheter-lock solutions may salvage precious catheters by eradicating biofilms. Staphylococcus epidermidis and Candida albicans are frequently isolated organisms in CRBSIs. We evaluated N-acetylcysteine (NAC), EDTA, ethanol and talactoferrin (TLF) individually and in combination with antibiotics against biofilms of S. epidermidis and C. albicans to identify effective catheter-lock solutions. Minimum biofilm-eradication concentrations causing 50% inhibition (MBEC(50)) for EDTA, NAC, ethanol and TLF were determined against biofilms of S. epidermidis and C. albicans formed on 96-well microtitre plates. Biomass, mean thickness and viability of S. epidermidis and C. albicans biofilms were evaluated after exposure to MBEC(50) concentrations of EDTA, NAC, ethanol and TLF. Antimicrobial combinations of EDTA, NAC, ethanol and TLF with nafcillin, vancomycin, fluconazole and amphotericin B were evaluated systematically for synergy using combination indices (CIs). EDTA, NAC, ethanol and TLF significantly reduced biofilm biomass and mean thickness (P<0.05, one-way ANOVA) of monomicrobial and polymicrobial biofilms as evaluated by confocal microscopy. CIs evaluated at equipotency ratios, and 50, 75 and 90 % effects, showed that EDTA, NAC, ethanol and TLF were synergistic (CI <1) with antibiotics (with few exceptions) against biofilms of S. epidermidis and C. albicans. EDTA, NAC, ethanol and TLF inhibit monomicrobial and polymicrobial biofilms of neonatal strains of S. epidermidis and C. albicans, and are synergistic with antibiotics. Catheter-lock solutions of EDTA, NAC and ethanol alone or in combination with antibiotics may be used to salvage infected catheters, which will directly impact on patient morbidity and health-care costs.