As a part of systematic investigation of synthesis, characterization and biological activity of several new substituted Tetrahydrothieno [2,3-c]pyridin-2-yl)urea derivatives. (3a-3k) have been synthesized by well known chemical reaction of one active methylene containing moiety with a carbonyl function and Sulfur powder to give 2- Aminothiophene ring system followed by derivatization of amino into urea by mixed anhydride method. The structures of all the synthesized compounds have been determined by their spectral and micro analytical data. All the synthesized products were evaluated for their antibacterial activity against Bacillus substilis, Escherichia coli. Klebsiella pneumoniae and Streptococcus aureus bacteria and antifungal activity against Aspergillus niger fungi respectively. Most of the synthesized compounds exhibited significant anti-bacterial and anti-fungal activities.