Diabetic wound (DW) is the most devastating complication resulting in significant mortality and morbidity in diabetic patients. The standard treatment of DW care fails to address the prerequisites of treating DW owing to its multifactorial pathophysiology. Henceforth, developing a single treatment strategy to handle all the loopholes may effectively manage DW. The objective of the current study was to formulate Human beta defensin-2 (HBD-2) loaded Poly (lactic-co-glycolic acid) (PLGA) nanoparticle impregnated in collagen/chitosan (COL-CS) composite scaffolds for the accelerated healing of DW. Upon investigation, the developed biodegradable crosslinked scaffold possesses low matrix degradation, optimum porosity, and sustained drug release than the non-crosslinked scaffold. In vitro studies revealed that the HBD-2 COL-CS scaffold was biocompatible and accelerated cell migration and angiogenesis. The HBD-2 COL-CS scaffold showed significant antimicrobial activity in S. aureus, E. coli, and P. aeruginosa. The in vivo studies revealed that the HBD-2 COL-CS treated group accelerated healing compared to those in COL-CS and control groups. The ELISA results indicated a significant decrease in MMP-9, TNF-α, MPO, NAG, and NO with an increase in IL-10 in HBD-2 COL-CS treated group. The accelerated healing in HBD-2 COL-CS treated group might be due to the synergistic effects of PLGA (collagen synthesis and deposition and positive angiogenic effect), HBD-2 (anti-inflammatory, antibacterial, positive angiogenic effect, cell proliferation, and migration), COL (established wound healer and stabilizer) and CS (antibacterial, controlled drug release).