IIn the present investigation, a new series of methyl 6-O-cinnamoyl-α-D-glucopyranoside derivatives were synthesized by the reaction of methyl α-D-glucopyranoside (1) with various non-traditional acylating agents in pyridine at -5°C. For this purpose, firstly, selective cinnamoylation of methyl α-D-glucopyranoside (1) has been carried out by the direct acylation method and afforded the methyl 6-O-cinnamoyl-α-D-glucopyranoside (2) in good yield. Secondly, in order to obtain newer products for antimicrobial screening studies, the 6-O-cinnamoyl derivative was further transformed to a series of 2,3,4-tri-O-acyl derivatives (4-14) containing a wide variety of functionalities in a single molecular framework. The structures of the newly synthesized compounds were characterized by IR, NMR and physicochemical properties analyses. All the compounds were evaluated for their in vitro antibacterial and antifungal activities using the disc diffusion and food poisoning methods. The study revealed that the compound methyl 6-O-cinnamoyl-2,3,4-tri-O-decanoyl-α-D-glucopyranoside (7) showed the highest inhibition activity against both Gram (+) B. subtilis and Gram (-) P. aeruginosa microorganisms. The tested compound methyl 6-O-cinnamoyl-2,3,4-tri-O-lauroyl-α-D-glucopyranoside (8) exhibited maximum mycelial growth inhibition against A. niger fungi. The acylated derivatives were found to be more effective against the fungal strains than those of the bacterial pathogens. This is the first report of the antimicrobial activity of the selected chemicals against the selected pathogens.