Background: Bacterial resistance to the available antibiotics is a life threatening issue and researchers are trying to find new drugs to overcome this problem. Amongst the different structural classes, thiazolidinone-4-one, as a new effective pharmacophore against various bacteria, has been introduced. Objective: A new series of 2-(5-(5-nitrothiophene-2-yl)-1,3,4-thiadiazole-2-ylimino)-5-arylidenethiazolidin- 4-one derivatives were designed and synthesized as new antibacterial agents. Method: Target compounds were synthesized during 5 steps and their in vitro antibacterial and anti-H. pylori activities were evaluated. The interaction of the most active derivatives with the probable targets was assessed by Auto Dock 4.2 Program. Results: The results showed that the most potent compounds, 18, 22 and 23, displayed antibacterial activity versus S.aureus, S.epidermidis, B.cereus and B.subtilis (MIC, 1.56-12.5 µg/mL) and none of the derivatives were active on tested Gram-negative bacteria. Compound 12 in all considered doses and compounds 10, and 27 had strong anti-H. pylori activity (inhibition zone >20 mm) in 25 μg disc. Docking studies determined suitable interactions and affinity of potent compounds with bacterial MUR B and H. pylori urease enzymes. Conclusion: According to the results most of the derivatives are effective anti-bacterial agents and docking evaluation confirmed their possible mechanisms of actions as MURB and Urease inhibitors.