Biofilms on biomaterial implants are hard to eradicate with antibiotics due to the protection offered by the biofilm mode of growth, especially when caused by antibiotic-resistant strains. Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used in various biomedical applications, such as targeted drug delivery and magnetic resonance imaging. Here, we evaluate the hypothesis that SPIONs can be effective in the treatment of biomaterial-associated infection. SPIONs can be targeted to the infection site using an external magnetic field, causing deep penetration in a biofilm and possibly effectiveness against antibiotic-resistant strains. We report that carboxyl-grafted SPIONs, magnetically concentrated in a biofilm, cause an approximately 8-fold higher percentage of dead staphylococci than does gentamicin for a gentamicin-resistant strain in a developing biofilm. Moreover, magnetically concentrated carboxyl-grafted SPIONs cause bacterial killing in an established biofilm. Thus magnetic targeting of SPIONs constitutes a promising alternative for the treatment of costly and recalcitrant biomaterial-associated infections by antibiotic-resistant strains.