Bacterial infections are a great threat to public health. The development of materials which prevent biofilm formation, and exhibit antibacterial properties, but are otherwise biocompatible, is a major research goal. In this study, a novel hybrid hydrogel with aggregation-induced emission (AIE) behavior was prepared through the self-assembly of commercial N-(9-fluorenylmethoxycarbonyl)-_L-phenylalanine (Fmoc-F) and berberine chloride (BBR). BBR self-assembled into nanoparticles which dispersed throughout the gel network, and endowed the hydrogel with AIE functionality. Fmoc-F/BBR hydrogel displayed enhanced antibacterial and anti-biofilm activity against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria when used as the active agent in the photodynamic therapy (PDT) process. Additionally, negligible cytotoxicity and low hemolytic activity were displayed. In vivo infection studies showed that Fmoc-F/BBR hydrogel facilitated the repair of wounds in rats infected with S. aureus. Therefore, Fmoc-F/BBR hydrogel provides potential applications in biomedicines, and especially in wound healing.