This work was aimed at synthesizing and characterizing urolithin B-encapsulated polyethyleneimine (PEI)-conjugated chitosan nanoparticles and their probable therapeutic use for diabetes-induced kidney damage. Nanoparticles with a specific formulation were prepared using the optimized formulation method, and various analyses were conducted on their properties. A completion of the conjugation between PEI and chitosan was identified through nuclear magnetic resonance (NMR) spectroscopy. The percent Encapsulation Efficiency (EE) along with Loading Efficiency (LE) were also determined and optimized to have the maximum encapsulation of the drug. The improved formulation of UB-PEI-CHI-NPs, with a particle size of 150 nm and a zeta potential of + 20.2 mV, achieved a percentage entrapment efficiency of 85.4%. Nanoparticle concentration ranging from 10 to 100 µg/mL resulted in cell survival rates above 85%. The in vitro drug release study revealed that urolithin B is released gradually over a longer duration. The MTT assay further ascertained the biocompatibility of the formulation and the cytotoxicity of the formulation in a dose-dependent manner. These outcomes indicate that urolithin-loaded PEI-conjugated chitosan nanoparticles might be employed as an effective therapeutic approach for the treatment of diabetic nephropathy, and hence, further in vivo experiments are required to test the prospects of the formulated nanoparticles.