A library of thiosemicarbazide derivatives of isoniazid 3-27, was synthesized and evaluated for their anti-inflammatory and urease inhibition activities, by using in vitro bioassays. Among these compounds 9, 10, 12, 21, and 26 were identified as new derivatives. Prolonged use of non-steroidal anti-inflammatory drugs (NSAIDs) and infections caused by Helicobacter pylori (ureolytic bacteria), are the two most significant causes of gastric and peptic ulcers. We focused on the identification of the dual inhibitors of inflammation and urease enzyme. Compound 23 was identified as the best dual inhibitor of inflammation (ROS; IC50 = 12.3 µg/mL), and urease enzyme inhibition activity (IC50 = 22.4 µM). Many of these compounds showed comparable activities to the standard anti-inflammatory drug (ibuprofen, IC50 = 11.2 µg/mL) and urease inhibitor (thiourea/acetohydraoxamic acid, IC50 = 21.1/20.3 µM). Compound 12 was found to be the most potent urease inhibitor (IC50 = 12.3 µM) and good inhibitor of inflammation (IC50 = 27.7 µg/mL). Compounds 19, 11, 13, 9, 17, 10, and 16, were also found to be potent inhibitors of urease. Cytotoxicity was also evaluated and all the compounds were found to be non-cytotoxic, except compound 18 and the parent drug isoniazid (IC50 = 29.5 and 28.5 µM, respectively).