Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three-dimensional structures with similar to 1?mu m resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen-sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stober method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (similar to 1?mu m). Two fluorophores, oxygen-sensitive Ru(Ph2phen3)Cl2, and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm. Biotechnol. Bioeng. 2012; 109: 26632670. (c) 2012 Wiley Periodicals, Inc.