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, 1H NMR, 13C-NMR, HMBC 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 moleculess containing -thioxo group are more potent than the -oxo group.