Carbohydrates are abundant naturally occurring biomolecules that play essential roles in a wide variety of biological processes. In this paper, we describe the synthesis of a series of methyl α-D-glucopyranoside (α-MGP) esters through regioselective acylation, by modification of the hydroxyl (-OH) group. Synthesized 6-O-acyl derivatives were further converted to 2,3,4-tri-O-acyl esters, in good yield. Structures of the newly designed α-MGP esters (termed compounds 1–8) were elucidated by spectroscopic, physicochemical, and elemental analyses. PASS (prediction of activity spectra for substances) and in vitro screening of compounds 1–8 against human and plant pathogenic microorganisms revealed substantial antibacterial and antifungal activities. Most of them showed >70% inhibition of fungal mycelial growth. In vitro effect of compound 7 against Ehrlich ascites carcinoma (EAC) cells, by MTT colorimetric assay, showed 23.97% of cell growth inhibition with an IC50 value of 1101.34 µg/ml. Molecular docking and nonbonding interaction studies were performed for three pathogen proteins Escherichia coli lectin FimH, Bacillus subtilis Obg GTP-binding protein, and Aspergillus flavus urate oxidase to evaluate binding affinities and interactions of compounds 1–8 with receptor proteins. A 100-ns molecular dynamics (MD) simulation study revealed the stable conformation and binding patterns/energy in a stimulating environment. In silico pharmacokinetics prediction and drug-likeness analysis of the compounds revealed improved kinetic parameters and satisfaction of all drug-likeness rules in regard to biological activity. Structure-activity relationship (SAR) studies, in combination with in vitro and in silico findings, indicated that among various acyl chains, lauroyl (C-12) and myristoyl (C-14), combined with α-MGP, were most effective against the tested bacterial and fungal pathogens. In conclusion, the designed α-MGP esters displayed enhanced pharmaceutical activities, and strong potential for development as antimicrobial agents.