Abstract: Glycosyl (iso) thioureas being an important intermediate in the synthesis of thioureido carbohydrates herein we reported the synthesis of per–O–acetyl lactosyl aryl isodithiobiuret derivatives. These compounds were synthesized by nucleophilic addition of per-O-acetyl lactosyl aryl isothiourea to tolyl isothiocyanate. Structures of these compounds were confirmed on the basis of IR, HNMR, Mass spectra and elemental analysis. These compounds were also screened for their in vitro antimicrobial activities against bacteria S. aureus, E. coli, P. vulgaris, P. aeruginosa and fungi A. niger, Penicillium.