A convenient microwave-assisted one-pot four-component synthetic approach was developed en route to novel functionalized benzo[a]pyridazino[3,4-c]phenazine derivatives starting from 2-hydroxy-1,4-naphthoquinone, aromatic aldehydes, methyl hydrazine and o-phenylenediamine. Nine new derivatives were successfully synthesized and subsequently evaluated in terms of their biological profiles. The results revealed good cytotoxic activities of compounds 6a, 6h against KB, HepG2, Lu1 and MCF7 human cancer cell lines. Besides that, compound 6d exhibited promising antimicrobial activities toward Staphylococcuc aureus and Bacillus subtilis bacterial strains with IC<sub>50</sub> < 6 μM.