Wounds associated with diabetes are difficult to heal and often stay unhealed, leading to higher morbidity and mortality in the diabetic population. A major barrier to the successful healing of chronic diabetic wounds is the presence of biofilm-mediated infections. In recent years, photodynamic therapy (PDT) has emerged as a promising treatment modality for the management of chronic wounds. The objective of the present study was to evaluate the effectiveness of toluidine blue- (TB-) loaded nanoparticle-mediated PDT in eradicating antibiotic-resistant polymicrobial biofilms ofPseudomonas aeruginosaand methicillin-resistantStaphylococcus aureusin anin vitromodel. Dioctyl sodium sulfosuccinate (aerosol OT, AOT)-alginate nanoparticles with high TB loading (10.8±2.2%) were formulated using a double emulsification cross-linking method. TB nanoparticles induced effective killing of planktonicP.aeruginosa(3.5 log10CFU) andS.aureus(>5 log10CFU) and their combined biofilms (2.8 log10CFU forP.aeruginosaversus 3.4 log10CFU forS.aureus). WhileP.aeruginosabiofilm was more resistant when compared to that ofS.aureus, our results demonstrated effective eradication of complex biofilms of dual bacterial strainsin vitro.