This study describes the synthesis and characterization of chlorambucil (CLB)-functionalized mesoporous silica nanoparticles (MSNs) for potential application in cancer therapy. The nanoparticles were designed with a diameter between 20 and 50 nm to optimize cellular uptake and avoid rapid clearance from the bloodstream. The synthesis method involved modifying a previously reported technique to reduce particle size. Successful functionalization with CLB was confirmed through various techniques, including Fourier transform infrared spectroscopy (FTIR) and elemental analysis. The cytotoxicity of the CLB-functionalized nanoparticles (MSN@NH2-CLB) was evaluated against human lung adenocarcinoma cells (A549) and colon carcinoma cells (CT26WT). The results suggest significantly higher cytotoxicity of MSN@NH2-CLB compared to unbound CLB, with improved selectivity towards cancer cells over normal cells. This suggests that MSN@NH2-CLB holds promise as a drug delivery system for targeted cancer therapy.