In search of effective antimicrobial and cytotoxic agents, a series of indole hybridized diazenyl derivatives ( to ) was efficiently prepared by condensation of diazotized -aminoacetophenone with indole or nitroindole followed by reaction with different aromatic/heteroaromatic amines of biological significance. The synthesized derivatives were characterized by various spectroscopic techniques.The antimicrobial evaluation of to was done by tube dilution method against various pathogenic bacterial and fungal strains. The active antimicrobial derivatives were further evaluated for cytotoxicity against human lung carcinoma cell line (HCT-116), breast cancer cell line (MDAMB231), leukemic cancer cell line (K562), and normal cell line (HEK293) by MTT assay using doxorubicin as the standard drug. The test derivatives were additionally docked for the B-subunit of enzyme DNA gyrase from at the ATPase binding site to study the molecular interactions using Schrodinger maestro software.Most of the synthesized derivatives have shown high activity against Gram-negative bacteria particularly and with MIC ranging from 1.95 to 7.81 μg/ml. The derivatives have demonstrated very less activity against tested Gram positive bacterial and fungal strains. The derivatives and have been found to active against breast cancer cell line and human colon carcinoma cell line having IC in the range of 19-65 µg/ml. All the derivatives were found to less potent against leukemic cancer cell line. The synthesized derivatives have revealed their safety by exhibiting very less cytotoxicity against the normal cell line (HEK-293) with IC > 100 µg/ml. Most of the active derivatives have shown good docking scores in comparison to the standard drugs against DNA gyrase from . Further ADME predictions by Qikprop module of the Schrodinger confirmed these molecules have drug like properties.The derivatives and have shown potential against Gram-negative bacteria and breast cancer cell line and can be used as a lead for rational drug designing of the antimicrobial and cytotoxic agents..