The process of wound healing and tissue regeneration involves several key mechanisms to ensure the production of new tissues with similar cellular functions. This study investigates the impact of pectin, a natural polysaccharide, and nebivolol hydrochloride (NBV), a nitric oxide (NO) donor drug, on wound healing. Utilizing ionotropic gelation, NBV-loaded pectin nanoparticles were developed following a 2231 full factorial design. The optimized formulation, determined using Design expert® software, exhibited an encapsulation efficiency percentage of 70.68%, zeta potential of −51.4 mV, and a particle size of 572 nm, characterized by a spherical, discrete morphology. An in vivo study was conducted to evaluate the effectiveness of the optimal formulation in wound healing compared to various controls. The results demonstrated the enhanced ability of the optimal formulation to accelerate wound healing. Moreover, histopathological examination further confirmed the formulation's benefits in tissue proliferation and collagen deposition at the wound site 15 days post-injury. This suggests that the developed formulation not only promotes faster healing but does so with minimal side effects, positioning it as a promising agent for effective wound healing and tissue regeneration.