Plaster of Paris, a well-known biomaterial, was synthesized from waste eggshells, which were chosen as an available bio-source of calcium. The produced plaster of Paris was characterized by X-ray diffraction (XRD), Fourier Transform Infrared (FTIR), Raman spectroscopy, UV-Vis spectroscopy, and SEM images along with a few crystallographic parameters such as crystallite size (Scherrer equation and different model equations), lattice parameters, crystallinity index, the volume of the unit cell, microstrain, dislocation density, growth preference, and residual stress from the XRD-sin2 Ψ technique. The biomedical competency of the prepared plaster of Paris was evaluated utilizing the cytotoxicity, hemolysis, and antimicrobial activity of E. coli and S. aureus. The cytotoxicity assessment has revealed that the percentages of viable cells were 93-98% and a highly hemocompatible nature (<5%) was exerted by the sample. The plaster of Paris only revealed antimicrobial properties against Gram-positive bacteria (S. aureus), and no effect was noticed for Gram-negative bacteria (E. coli).