Antimicrobial photodynamic therapy (PDT) is used for the eradication of pathogenic microbial cells and involves the light-excitation of dyes in the presence of O2 yielding reactive oxygen species including the hydroxyl radical (OH) and singlet oxygen ((1)O2). In order to chemically enhance PDT by the formation of longer-lived radical species, we asked whether thiocyanate (SCN(-)) could potentiate the methylene blue (MB) and light mediated killing of the Gram-positive Staphylococcus aureus and the Gram-negative Escherichia coli. SCN(-) enhanced PDT (10µM MB, 5J/cm(2) 660nmhv) killing in a concentration dependent manner of S. aureus by 2.5 log10 to a maximum of 4.2 log10 at 10mM (p<0.001) and increased killing of E. coli by 3.6 log10 to a maximum of 5.0 log10 at 10mM (p<0.01). We determined that SCN(-) rapidly depleted O2 from an irradiated MB system, reacting exclusively with (1)O2, without quenching the MB excited triplet state. SCN(-) reacted with (1)O2, producing the sulfur trioxide radical anion (a sulfur-centered radical demonstrated by EPR spin-trapping). We found that MB-PDT of SCN(-) in solution produced both sulfite and cyanide anions, and that addition of each of these salts separately enhanced MB-PDT killing of bacteria. We were unable to detect EPR signals of OH, which, together with kinetic data, strongly suggests that MB, known to produce OH and (1)O2, may, under the conditions used, preferentially form (1)O2.