The aim of this study is to synthesize and investigate antimicrobial and cytotoxic activity of 3 H-quinazolinone (1), and its derivatives, which are 3-(4-hydroxyphenyl)-3,4-dihydroquinazolin-4-one(2),3-(3-hydroxyphenyl)-3,4-dihydroquinazolin-4-one(3), 3-(pyridin-3-ylmethy)-3,4-dihydroquinazolin-4-one(4) and derivatives of 2-methyl-3H-quinazolinone, which are 2-methyl-3-phenyl-3,4-dihydroquinazolin-4-one(6), 2-methyl-3(4-hydroxyphenyl)-3,4- dihydroquinazolin-4-one(7),2-methyl-3(4-nitrophenyl)-3,4-dihydroquinazolin-4-one(8), compounds. Compounds 2 and 3 were synthesized newly. All compounds were characterized via infrared, 1H-NMR, 13 C-NMR, elemental and mass spectral analysis. The compounds were screened for their antimicrobial activity in vitro against E. faecalis ATCC 29212, P. aeruginosa ATCC 27853, S. aureus ATCC 29213, E. Coli ATCC 25922 bacteria and compared with commercial antibiotics (Ampicillin and Gentamicin). The determination of the antimicrobial activity was done by using the broth microdilution methods. The antimicrobial test results indicated that the compounds (1, 4, 6, and 8) have MIC values against P. aeruginosa ATCC 27853 lower than ampicillin. But clear cytotoxic effects on proliferation of the gingival fibroblasts applications were observed in 1, 4, 6, and 8 when compared to the control group.