This research aims to introduce a new wound dressing with antibacterial and anti-inflammatory properties made from chitosan and copper-containing Janus nanoparticles (JNPs). The JNPs were synthesized by attaching copper to PDA nanospheres, which were then embedded in Chitosan at different concentrations. The resulting spherical JNPs had a mean size of 208 ± 96 nm, and EDX mapping showed successful adhesion of Cu2+ ions to PDA nanospheres with a total Cu2+ content of 16.5 wt%. The samples exhibited interconnected porous structures, increasing JNPs concentration resulting in larger pore size and higher porosity. The addition of JNPs to 10 % (Ch-JNP 10) resulted in the highest strength, young modulus, and crystallinity, while a reverse trend was observed at higher JNPs content. JNPs improve the antibacterial activity of chitosan-based dressing, especially against E. coli. All samples were biocompatible and did not exhibit any cytotoxic effects. Ch-JNP10 had higher cellular density, confluency, and collagen secretion than other samples. The in vivo study demonstrated that Ch-JNP10 induced epithelialization and oriented collagen fiber formation while reducing inflammation. Overall, Ch-JNP10 may be a potential wound dressing for chronic wounds.