Creating an efficient antimicrobial photodynamic therapy (PDT) method while reducing the difficulty of preparation and operation is much anticipated but challenging. In this research, we facilely developed glycol chitosan (GC)/protoporphyrin IX (PpIX) nano-micelles for effective photodynamic inactivation of Gram-positive bacteria. The mixture of glycol chitosan (GC) and protoporphyrin IX (PpIX) self-assembled into positively charged GC/PpIX nano-micelles through hydrophobic interaction and electrostatic interaction. GC/PpIX nano-micelles significantly improved singlet oxygen generation, and notably enhanced the antibacterial PDT effect toward Gram-positive bacteria in a preincubation-free manner without developing drug resistance. Upon binding to the bacterial cells through electrostatic interaction, GC/PpIX nano-micelles were disassembled, leaving GC on the bacterial surface, inducing bacterial cell aggregation and acting like a glue to coat the bacterial surface and release PpIX into the cells, generating singlet oxygen under light irradiation to impair DNA. Collectively, the easy construction of GC/PpIX nano-micelles offers a modality for effective antibacterial PDT with simple operation and without drug resistance development, benefiting the treatment of Gram-positive bacterial infections in clinic.