The growing risk of antimicrobial resistance besides the continuous increase in the number of cancer patients represents a great threat to global health, which requires intensified efforts to discover new bioactive compounds to use as antimicrobial and anticancer agents. Thus, a new set of pyridothienopyrimidine derivatives was synthesized via cyclization reactions of 3-amino-thieno[2,3-]pyridine-2-carboxamides with different reagents. All new compounds were evaluated against five bacterial and five fungal strains. Many of the target compounds showed significant antimicrobial activity. In addition, the new derivatives were further subjected to cytotoxicity evaluation against HepG-2 and MCF-7 cancer cell lines. The most potent cytotoxic candidates (, , , , and ) were examined as EGFR kinase inhibitors. Molecular docking study was also performed to explore the binding modes of these derivatives at the active site of EGFR-PK. Compounds , and displayed broad spectrum antimicrobial activity with MIC ranges of 4-16 µg/mL and potent cytotoxic activity with IC ranges of 1.17-2.79 µM. In addition, they provided suppressing activity against EGFR with IC ranges of 7.27-17.29 nM, higher than that of erlotinib, IC = 27.01 nM.