Diabetic foot ulcers (DFUs) are one of the major complications of diabetes mellitus and the principal cause of lower limb amputations. Antimicrobial photodynamic therapy (aPDT) is a promising alternative therapeutic strategy because it is not limited by drug resistance. A new photosensitizer – a low molecular weight dicationic imidazolyl chlorin – was developed for aPDT to improve permeation in biofilms and absorption in the phototherapeutic window. The efficacy of aPDT with IC-H-Me2+ was tested in biofilms from clinical isolates from patients with DFUs both alone and when potentiated by potassium iodide (KI). aPDT was performed using 1µM IC-H-Me2+ and 50 mM KI with one-hour incubation period, followed by exposure to a light dose of 5 J/cm2 using a 660 nm LED. The fifteen different clinical isolates of MRSA, MSSA, S. epidermidis and Pseudomonas aeruginosa produced different levels of biofilm when growing for the same period of time (9 logs CFU to 20 logs CFU). The susceptibility of biofilms to respond to aPDT depends on the amount of biofilm and the more mature biofilms tend to have a poorer response to treatment. Nevertheless, all biofilms showed more than 3 logs CFU inactivation when aPDT was performed with 1µM IC-H-Me2+ and light, and most of them where eradicated when aPDT was potentiated by the addition of 50 mM KI. IC-H-Me2+ has excellent physical and photochemical properties as a photosensitizer for aPDT. Our results offer the basis for the development of formulations that can selectively eradicate biofilms established in infected wounds using aPDT.