A series of novel substituted hydrazono indolo[2,1-b]quinazoline-6,12-dione analogues have been synthesized and screened for their in vitro cytotoxic and antimicrobial activities. Among all the target compounds, 3c exhibited the most potent inhibitory activity against three cancer cell lines MCF-7, A549, HeLa with IC50 values 07.14±1.285μM, 09.18±0.968μM and 10.57±0.581μM respectively, while maintaining low toxicity towards non-cancer originated cell line, HEK-293. The detailed studies about molecular interactions with probable target protein indoleamine 2,3-dioxygenase (IDO1) were done by using docking simulations. The results from docking models are in consistent with the experimental in vitro cytotoxic activity conclusions i.e. 3c shows the highest binding energy -11.25kcal/mol. Furthermore, antimicrobial studies revealed that the compound 3e has shown excellent anti bacterial activity against four tested strains and the compounds 3b, 3e and 3f have shown good anti fungal activity against two tested organisms as compared with their standard drugs.