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To determine the effect of a composition comprising ovotransferrin (OT), protamine sulfate (PS) and ethylenediaminetetraacetic acid (EDTA) on biofilm formation by catheter-associated bacteria.The in vitro activity of OT, PS and EDTA alone and in combinations against biofilm formation by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Enterococcus faecalis and Staphylococcus epidermidis was investigated. All the three compounds either alone or in combinations failed to inhibit the growth completely at the concentrations tested. However, the subinhibitory concentrations of three compounds in a composition showed synergistic inhibitory effect on biofilm formation by K. pneumoniae, Ps. aeruginosa and S. epidermidis. Furthermore, 79–95% reduction in Ps. aeruginosa and S. epidermidis biofilm formation was observed in a clear vinyl urinary catheter treated with the composition.The subinhibitory concentrations of OT, PS and EDTA in a composition were effective in reducing biofilm formation by catheter-associated bacteria.This study shows that a synergistic composition-comprising non-antibiotic generally regarded as safe (GRAS) compounds such as OT, PS and EDTA may be used in the prevention of catheter-related infections.