Some fused  Pyrano[2,3-b]Pyridines derivatives have been synthesized by the cyclo condensation reaction of amino cyano spiro pyrano derivative (3'-Acetyl-6'-amino-2'-methyl-2-oxospiro[indoline-3,4'- pyran]-5'-carbonitrile) with some arylidene malononitrile derivatives. The synthesized compounds have been characterized by FT-IR, NMR spectroscopy, and elemental analyses.  Newly synthesized compounds were evaluated for antimicrobial activity against filamentous and unicellular fungi Aspergillus fumigatus and Candida albicans, gram-negative bacteria Pseudomonas aeruginosa, and gram-positive bacteria Staphylococcus aureus. The synthesized compounds promise to possess high selectivity and limited side effects.  All compounds exhibited effective to moderate action against all tested microorganisms thanks to spiro heterocycles, which have a vital part in biological processes, pharmacological and therapeutic activities, and a combination of Pyran, isatin, and pyridine moieties which exhibit fungicidal, antibacterial properties, and unique, versatile molecular structure. Compound 8 was the most potent and active compound against the tested bacteria; particularly, against Staphylococcus aureus as a gram-positive bacteria with an impact (up to 90% ).