Background: Mango seed kernels, an agro-industrial waste byproduct, constitute approximately 40–50% of the fruit’s weight and serve as a substantial source of starch. There are only a few reported studies on the pharmaceutical applications of Mango Seed Kernel Starch (MSKS) and drug carriers produced from this source. This study aims to isolate starch from mango seed kernels (MSKS), prepare drug-loaded mango seed kernel starch nanoparticles (MSKSNPs), and study the in vitro transdermal permeation. Methods: The MSKS was prepared using the alkaline method and freeze-dried. The prepared starch was analyzed for physicochemical properties relative to corn starch. The mango seed kernel starch nanoparticles (MSKSNPs) were prepared using mild alkali hydrolysis and the ultrasonication method. The model drug selected for this study was diclofenac sodium (DS), a commonly prescribed non-steroidal anti-inflammatory drug. Results: The average particle size of the drug-loaded nanoparticles was 140.0 ± 3.6 nm, with a PDI of 0.42 ± 0.03. The Transmission Electron Microscopy images confirmed the globular structure of MSKSNPs. X-ray Diffraction revealed that the diclofenac crystal size decreased to 14 nm from 33 nm in the pure drug, confirming the amorphous nature of MSKSNPs. The drug-loaded MSKSNPs showed a % encapsulation efficiency of 92.4 ± 3.7 and % drug loading of 31.08 ± 0.96. The cumulative drug released from MSKSNPs after 6 h, 12 h, and 24 h was found to be 25.58 ± 1.30, 59.68 ± 2.98, and 127.5 ± 6.4 μg/cm^2, respectively, which was more than the ethanolic drug solution with statistical significance (p-value < 0.01) along with enhanced skin retention. Conclusions: MSKSNPs were efficiently synthesized using mild alkali hydrolysis and ultrasonication, showing enhanced transdermal delivery. Skin retention was significantly higher in MSKSNPs (p-value < 0.05). The cytotoxic studies revealed that both formulations exhibit similar dose-dependent cytotoxicity, with no significant difference (p > 0.05) in their potency under the tested conditions.