A series of novel thiourea analogs of 3,4-dihydropyrimidin-2(1-H)-one derivatives of biological interest were prepared by sequential Bigineli's reaction, reduction followed by reaction of resulting amine with different arylisothiocynates. All the synthesized compounds (1–23) were screened against the proinflammatory cytokines (TNF-a and IL-6) and antimicrobial activity (antibacterial and antifungal). Biological data revealed that among all the compounds screened, compounds 6, 10, 11, 12, 13, 14, 17, and 20 found to have promising anti-inflammatory activity (42–78% TNF-a and 54–96% IL-6 inhibitory activity at 10 lM). Remarkably, compounds 3, 4, 5, 6, 15, 22, and 23 revealed promising antimicrobial activity at minimum inhibitory concentration of 10–40 lg/ml against selected pathogenic bacteria and fungi.