A series of novel phenothiazine derivatives with biologically active moieties were synthesized. Treatment of 3-oxo-3-(10H-phenothiazin-10-yl)propanenitrile (2) with carbon disulfide gave 3,3-dimercapto-2-(10H-phenothiazine-10-carbonyl)acrylonitrile (4) which reacted with different reagents to afford novel fused heterocyclic compounds. The newly synthesized compounds were examined for their antimicrobial activity. Among the tested compounds, compounds 2, 4, 5a,b and 13 showed the highest antimicrobial activity. Molecular modeling was evaluated and was in agreement with experimental biological activity results.