Bis-thiobarbiturate derivatives 1-15 have been synthesized and evaluated for their in vitro β-glucuronidase inhibitory potential. The structures of all compounds were confirmed through spectroscopic techniques such as EI-MS and 1HNMR. Compounds 13 (IC50 = 29.42 ± 0.61µM) showed potent β-glucuronidase inhibitory potential better than the standard inhibitor (D-saccharic acid 1, 4 lactone, IC50 = 48.4 ± 1.25 µM). The compounds 2 (IC50 = 48.45 ± 0.39 µM), 3 (IC50 = 53.12 ± 0.22 µM), 4 (IC50 = 55.12 ± 1.13µM) and 9 (IC50 = 49.84 ±1.46µM) showed b-glucuronidase inhibitory potential comparable to the standard inhibitor. Compounds 1 (IC50 = 68.45 ± 0.33 µM), 5 (IC50 = 61.18 ± 1.42µM), 6 (IC50 = 92.57 ±2.80 µM), 7 (IC50 = 61.27 ± 0.45 µM), 8 (IC50 = 62.85 ± 0.79µM), and 12 (IC50 = 77.56 ± 1.32µM) also showed good inhibitory potential. The remaining compounds are found to be completely inactive. The structure-activity relationship was established for these compounds. This study identified a novel class of β-glucuronidase inhibitors.