Bio-scaffolds, while mimicking the morphology of native tissue and demonstrating suitable mechanical strength, enhanced cell adhesion, proliferation, infiltration, and differentiation, are often prone to failure due to microbial infections. As a result, tissue engineers are seeking ideal scaffolds with antibacterial properties. In this study, silver nanoparticles (AgNPs) were integrated into cotton-like polyacrylonitrile nanofibers via a polydopamine (PDA) interlayer (Ag@p-PAN). These Ag@p-PAN nanofibers were then incorporated into the chitosan (CS) matrix, developing an antibacterial CS/Ag@p-PAN composite scaffold. The composite scaffold features an interconnected porous morphology with fiber-infused pore walls, improved water absorption and swelling properties, a controlled degradation profile, enhanced porosity, better mechanical strength, strong antibacterial properties, and excellent MC3T3-E1 cell viability, adhesion, proliferation, and infiltration. This study presents a novel method for reinforcing CS-based scaffolds by incorporating bioactive nanofibers, offering potential applications in tissue engineering and other biomedical fields.