and are opportunistic pathogens that cause life-threatening infections. This study focused on using photodynamic inactivation (PDI) to eliminate mixed biofilms of formed on poly (urethane) (PU) discs functionalized with a nanocomposite layer containing phloxine B (PhB). Additionally, the effect of PDI on the and genes of was examined in mixed biofilms. Spectral analysis showed a continuous release of PhB from the nanocomposite in Mueller-Hinton broth within 48 h, with a released amount of PhB < 5% of the total amount. The anti-biofilm effectiveness of the light-activated nanocomposite with PhB showed a reduction in the survival rate of biofilm cells to 0.35% and 31.79% for and , respectively, compared to the control biofilm on PU alone. Scanning electron microscopy images showed that the nanocomposite effectively reduced the colonization and growth of the mixed biofilm. While PDI reduced the regulation of the gene, the gene was upregulated. Nevertheless, the cell survival of the - biofilm was significantly reduced, showing great potential for the elimination of mixed biofilms.