A new comparative series of barbituric acid and thiobarbituric acid based chalcone derivatives bearing the pyrimidine nucleus were synthesized. The chemical structures of the resulting molecules were characterised by means of FT-IR (Fourier Transform Infrared) 1 H NMR, 13 C-NMR (Nuclear Magnetic Resonance) and HMBC (Heteronuclear Multiple-Bond Correlation) and Elemental Analysis. All synthesized compounds were subjected to in vitro antimicrobial screening against four bacterial strains i.e., one Gram Positive (Bacillus subtilis MTCC 441), two Gram Negative (E. coli MTCC 443, P. aeruginosa MTCC 1688), and one Fungal (C. albicans MTCC 227) Strains. The structure activity relationship is discussed on the basis of bioactivity results, various functional groups present and position of the functional group at various positions of the synthesized compounds. The comparative antimicrobial activity study of both of the series elucidated that shows the chalcone compounds containing-thioketo group are more potent than the-keto group. very versatile as physiologically active compounds and substrates for the evaluation of various organic syntheses. So, in light of above facts of pyrimidine, barbituric acid, thiobarbituric acid and chalcones, we continue our earlier work on synthesis of 5-acetyl barbituric acid 4 and 5-acetyl thiobarbituric acid 4' based chalcones 5 (a-k) and 5 (a'-k'). All the analogs were screened for their antimicrobial activity and their comparative results are discussed with respect to one Gram Positive (Bacillus subtilis MTCC 441), two Gram Negative (E. coli MTCC 443, P. aeruginosa MTCC 1688), and one Fungal (C. albicans MTCC 227) Species and effects of functional groups and position of functional groups on various microbial strains. Experimental Methods Chemicals and solvents were obtained from commercial sources and used as received throughout the investigation. Melting points were determined in open capillaries on a Veego electronic apparatus VMP-D (Veego Instrument Corporation, Mumbai, India) and are uncorrected. IR spectra (4000-400 cm-1) of synthesized compounds were recorded on a Perkin Elmer-Spectrum RX-IFTIR spectrophotometer using KBr pellets. Thin layer chromatography was performed on object glass slides (2 × 7.5 cm) coated with silica gel-G and spots were visualized under UV irradiation. 1 H NMR and 13 C NMR spectra were recorded on an Advance-II (Bruker) model using DMSO as a solvent and TMS as internal standard with 1 H resonant frequency of 400 MHz and 13 C resonant frequency of 100 MHz. The 1 H NMR and 13 C NMR chemical shifts were reported as parts per million (ppm) downfield from TMS