24 Treatment of Staphylococcus aureus biofilm-related infections represents an important 25 medical challenge worldwide, as biofilms, even of drug-susceptible S. aureus strains, are 26 highly refectory to conventional antibiotic therapy. Nitroxides were recently shown to induce 27 dispersal of Gram-negative biofilms in vitro, but their action against Gram-positive bacterial 28 biofilms remains unknown. Here we demonstrate that the biofilm dispersal activity of 29 nitroxides extends to S. aureus, a clinically important Gram-positive pathogen. Co-30 administration of the nitroxide CTEMPO with ciprofloxacin significantly improved the 31 antibiotic's biofilm-eradication activity against S. aureus. Moreover, covalently linking the 32 nitroxide to the antibiotic moiety further reduced ciprofloxacin's minimal biofilm eradication 33 concentration. Microscopy analysis revealed that fluorescent nitroxide-antibiotic hybrids 34 could penetrate S. aureus biofilms and enter into cells localising at the surface and base of 35 the biofilm structure. No toxicity was observed for the nitroxide CTEMPO and the nitroxide-36 antibiotic hybrids against human cells. Taken together, our results show that nitroxides can 37 mediate dispersal of Gram-positive biofilms and that dual-acting biofilm-eradication 38 antibiotics could provide broad-spectrum therapies for the treatment of biofilm-related 39 infections.40