Methyl α-D-glucopyranoside and its seven acylated derivatives have been used to develop antibacterial and antifungal drugs by using in silico and in vitro antimicrobial functionality tests against five pathogenic bacteria and two fungi. Methyl α-D- glucopyranoside derivatives (1-8) were synthesized, purified, and characterized by physicochemical, elemental, and spectroscopic methods. Compounds 3 (zone of inhibition, 22±0.3 mm) and 8 (zone of inhibition, 24±0.4 mm) showed the highest inhibition against Bacillus subtilis and Staphylococcus aureus. A MIC value of 0.275±0.01 mg/ml was found for derivative 8 against S. ebony whereas the MBC value recorded for derivative 3 against S. aureus was 1.70±0.01 mg/ml. Most of these derivatives showed >78% inhibition of fungal mycelial growth. The in vitro effect of compound 8 against Ehrlich ascites carcinoma (EAC) cells, by MTT colorimetric assay, showed 25.97% of cell growth inhibition with an IC50 value of 1024.83 μg/ml. A DFT technique was used to determine the Highest Occupied Molecular Orbital (HOMO) and Lowest Unoccupied Molecular Orbital (LUMO), and the energy gap between them. Furthermore, the chemical reactivity and global descriptors were computed from the HOMO and LUMO values. The most crucial aspect of this research is the molecular docking against two gram-positive bacterial proteins (B. subtilis and S. aureus), two gram-negative bacterial proteins (Escherichia coli and Pseudomonas aeruginosa), and six fungal proteins (Aspergillus niger, Aspergillus flavus, Rhizomucor miehei, Mucor lusitanicus, Candida albicans and Candida Auris). In most cases, docking scores crossed the scores of the standard drugs, Azithromycin and Nystatin. A 100-ns molecular dynamics (MD) simulation study revealed stable conformation and binding patterns/energy in a stimulating environment. The range of quantitative structure-activity relationship (QSAR) and pIC50 found was between 4.19–9.15, signifying these compounds to be physiologically effective towards microbes. Most importantly, these compounds are non–carcinogenic, have low toxicity in aquatic and non–aquatic species, and are highly soluble in water and stable, indicating the suitability of these compounds as antimicrobial agents for therapeutic and drug development purposes.