The superfine powder of black pepper at nanometeric scale was synthesized using High Energy Ball Milling equipment. Current research study shows alteration in crystal structure and surface morphology due to grinding at different milling time. The Electron microscopy studies revealed that the superfine Nano powder is less than 50 nm in size and surface structure was found to change. The microstructure accessibility clearly specifies tiny nanoparticles development, which is observed as an agglomerated structure. FTIR and UV-vis-NIR spectrum shows that the internal covalent nature of materials did not change due to milling, but surface reactivity was found changed, which was further confirmed by Scanning Electron Microscope. The optical properties were highlighted by the observed changes in the colour which has changed from dark brown to light brown confirming the changes in properties in pretext to the crystal structure of the synthesized black pepper nano powder. The effect of the prepared Nano formulations was tested on the cell health index of the isolated mice splenocytes, which was done by using MTT assay. The result of the assay showcased reduced cell toxicity and increased cell viability due to change in crystal structure of superfine powder. The present research reveals that nanometric food particles can remarkably enhance the physicochemical properties, which are useful for its applications in agriculture, food, Biomedical science as new functional food materials.