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; IC
50
= 12.3 µg/mL), and urease enzyme inhibition activity (IC
50
= 22.4 µM). Many of these compounds showed comparable activities to the standard anti-inflammatory drug (ibuprofen, IC
50
= 11.2 µg/mL) and urease inhibitor (thiourea/acetohydraoxamic acid, IC
50
= 21.1/20.3 µM). Compound
12
was found to be the most potent urease inhibitor (IC
50
= 12.3 µM) and good inhibitor of inflammation (IC
50
= 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 (IC
50
= 29.5 and 28.5 µM, respectively).