A new series of chalcone analogs namely 5-(3-phenyl-acryloyl)-2-thioxo-dihydro-pyrimidine-4,6-dione have been synthesized from the key intermediate 5-acetyl-2-thioxo-dihydro-pyrimidine-4,6-dione 40 with different aldehyde derivatives were performed to get the target compounds as thiobarbituric acid-based chalcones 5(a0 –k0 ) and they were obtained in excellent yields. The newly synthesized compounds were characterized by spectral analysis (FT-IR, 1 H NMR, 13C NMR, and UV spectroscopy) and elemental analysis. The synthesized compounds were evaluated for their antimicrobial activity against five bacterial strains (S. aureus, S. pyogenes, E. coli, K. pneumoniae, and P. aeruginosa) and four fungal strains (C. albicans, A. clavatus, T. rubrum, and Penicillium wild strain). Among the screened compounds, 5e0 and f 0 showed comparable activity (minimum inhibitory concentration = 500 lg/mL) nearly to that of standard antibiotics griseofulvin.