A series of novel chromeno [4,3] pyrimidine-2(5H)-thione derivatives were synthesized and characterized by FT-IR, 1H-NMR, and Mass spectroscopy analysis with the aim of developing potent antimicrobial agents. The paper disc diffusion method and agar streak dilution method were performed for screening in vitro antimicrobial activity and the results are represented as a zone of inhibition and MIC, respectively. All compounds exhibited weak to potent anti-microbial activity against the tested microorganism such as B. subtilis, E. coli, A. niger & P. chrysogenum. The relationship between the functional group variation and the antimicrobial activity of the evaluated compounds was discussed. Out of various synthesized compounds, 3-(1-(dimethylamino)-2-(4-hydroxyphenyl) vinyl)-9-fluoro-5-hydroxy-3H-chromeno [4,3] pyrimidine-2(5H)-thione (C1) was found to be the most active compound. Keywords: Pyrimidine, Chromene, Antimicrobial activity, Zone of inhibition, MIC