Antimicrobial resistance (AMR) represents a critical global health challenge, driving the need for innovative therapeutic strategies. This study introduces self-assembled nanoparticles based on fourth-generation polyamidoamine (PAMAM-G4) dendrimers and Phloxine B (PhB), forming G4-PhB nanoparticles as an advanced platform for antimicrobial photodynamic therapy (aPDT). The optimal dendrimer:dye molar ratio was determined through dynamic light scattering (DLS) titration experiments, yielding a 1:15 G4:PhB ratio. The resulting G4-PhB nanoparticles were spherical, with a hydrodynamic diameter of 260 +/- 15 nm, a narrow polydispersity index (PDI) of 0.264 +/- 0.085, and a positive zeta potential of 8.71 +/- 2.88 mV, indicating monodispersity and colloidal stability. These features were corroborated by morphological analyses using TEM and AFM. Cytotoxicity assays conducted on murine fibroblasts (3 T3 cell line), using MTT, neutral red uptake, and crystal violet staining revealed that G4-PhB nanoparticles are intrinsically non-toxic, contrasting with the EDTA-PhB complex, which exhibited significant cytotoxic effects. Antibacterial activity was evaluated against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). While free PhB demonstrated bactericidal effects exclusively against SA, the G4-PhB nanoparticles exhibited enhanced activity against both bacterial strains, notably overcoming the limitations of free PhB against PA. These findings highlight the versatility and effectiveness of G4-PhB nanoparticles as a biocompatible and non-invasive system for localized aPDT, with potential applications in wound healing for immunocompromised patients. This work provides a robust foundation for future research into dendrimer-based photosensitizers as innovative solutions to pressing biomedical challenges.