Pure and glycine-doped strontium formate dihydrate [Sr(HCOO)₂·2H₂O] single crystals were grown by slow evaporation using glycine concentrations of 0.005 – 0.1 M. The influence of glycine incorporation on structural, vibrational, electronic, and antimicrobial properties was studied experimentally and using DFT (B3LYP/6-311G**). The HOMO – LUMO gap (7.111 eV) indicates stability, while the hyperpolarizability (6973.787 a.u.) suggests potential nonlinear optical behavior. FT-IR and FT-Raman analyses show characteristic modes with slight shifts due to glycine–lattice interactions. Doped crystals exhibit enhanced antimicrobial activity, with a maximum inhibition zone of 36 mm against Escherichia coli at 0.1 M concentration. Results indicate that glycine affects the electronic and biological properties of strontium formate dihydrate, suggesting potential optical and antimicrobial applications.