The aim of this work is to synthesize, characterize and evaluate the biological activity of 1,4-naphthoquinone derivatives ligands and their metal—Co(II), Ni(II) and Cu(II) chelates. Continuing our work with another derivatives of 1,4-naphthoquinone ligands, this work had been constructed for synthesis of new ligands derived from 1,4-naphthoquinone such as 3-thioacetyl-2-amino-1,4-naphthoquinone and 2-benzoyl-3-amino-1,4-naphthoquinone (L¹-L²) which characterized on the basis of elemental analysis, electronic, IR, mass, ¹H-NMR spectral data. The synthesized ligands have been carried out to achieve the coordination behavior towards bi-valent metal ions like cobalt, nickel and copper. The solid chelates of the different ligands were prepared and subjected to analytical techniques such as elemental analyses, spectroscopic techniques including mass, ¹H-NMR, and IR spectroscopy, and thermal analyses techniques. The chelates were found to have octahedral geometry. The biological activity of the prepared ligands and their binary metals complexes were also screened against different antifungal and antibacterial organisms.