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To investigate the ability of a mixture of phage K and six of its modified derivatives to prevent biofilm formation by Staphylococcus aureus and also to reduce the established biofilm density.The bioluminescence-producing Staph. aureus Xen29 strain was used in the study, and incubation of this strain in static microtitre plates at 37°C for 48 h confirmed its strong biofilm-forming capacity. Subsequently, removal of established biofilms of Staph. aureus Xen29 with the high-titre phage combination was investigated over time periods of 24 h, 48 h and 72 h. Results suggested that these biofilms were eliminated in a time-dependant manner, with biofilm biomass reduction significantly greater after 72 h than after 24–48 h. In addition, initial challenge of Staph. aureus Xen29 with the phage cocktail resulted in the complete inhibition of biofilm formation over a 48-h period with no appearance of phage resistance.In general, our findings demonstrate the potential use of a modified phage combination for the prevention and successful treatment of Staph. aureus biofilms, which are implicated in several antibiotic-resistant infections.This study highlights the first use of phage K for the successful removal and prevention of biofilms of Staph. aureus.