A new strategy for designing and assembling a novel class of functionalized pyridine-based benzothiazole and benzimidazole incorporating sulfonamide moieties was developed. The synthesis was carried out by reacting N-cyanoacetoarylsulfonylhydrazide with various electrophiles such as 2-(benzo[d]thiazol-2-yl)-3,3-bis(alkylthio)acrylonitriles and 2-(benzo[d]imidazol-2-yl)-3,3-bis(methylthio)-acrylonitriles, as well as 2-ethoxyl acrylonitrile derivatives. The synthesized compounds were tested for their antiviral and antimicrobial potency. Two of the synthesized compounds, 15c and 15d, showed more than 50% viral reduction against HSV-1 and CBV4, with significant IC50 and CC50 values. The two potent compounds 15c and 15d have also shown inhibitory activity against Hsp90α protein with IC50 values of 10.24 and 4.48 μg/mL, respectively. A combination of 15c and 15d with acyclovir has led to IC50 values that are lower than that of acyclovir alone. Molecular modeling studies were used to identify the interactions between the 15c and 15d compounds and the active site of Hsp90α enzyme. The antimicrobial investigation of the new compounds has also shown that 8b and 15d exhibited a higher inhibition zone (IZ) than sulfadiazine and gentamicin against Klebsiella pneumonia, whereas 9a showed higher IZ than ampicillin against Staphylococcus aureus. According to the enzyme assay study on dihydrofolate reductase, 9a was shown to be the most potent compound among all examined compounds.