A novel, quick, environmentally safe, and one-pot synthesis of a series of N,N-bis(cyanoacetyl)hydrazine derivatives, bis-imino-2H-chromenes and bis-2-oxo-2H-chromene derivatives have been designed. Some selected newly synthesized compounds were investigated in vitro for their antibacterial activity. Compound 5j is the most toxic compound against Staphylococcus aureus with activity index 171%, followed by compound 15b with activity index 136% compared to standard drug ampicillin. Moreover, compound 15a is the most toxic compound against Escherichia coli with activity index 111% compared to standard drug gentamicin. Minimum inhibitory concentration (MIC) was carried out for compounds with high antibacterial activity. Compound 5j has good MIC (7.8μg/ml) against Staphylococcus aureus while 15a has good MIC (31.25μg/ml) against Streptococcus mutans which is better than MIC of the standard drug ampicillin (MIC=62.5μg/ml). Compounds 5j, 5k, 15a, 15b and 15e which have good MIC values were introduced to enzyme assay against DNA gyrase and topoisomerase IV. The results showed that compound 15a can strongly inhibit DNA gyrase and topoisomerase IV (IC50=27.30 and 25.52μM respectively), compared to methotrexate as the standard drug (IC50=29.01 and 23.55μM respectively). Structure-activity relationships were also discussed based on the biological and docking simulation results.