Ampicillinacetylacetone Schiff base (HAA) and its complexes with Co(II), Ni(II), Cu(II), Eu(III) and Gd(III) obtained in pure form. The obtained compounds characterized by CHNM%, FTIR, UV–Vis, Fluorescence, 1HNMR, Mass spectra, DTA, TGA, Magnetic susceptibility, X-ray, AAS, melting point, solubility in different solvents and the conductivity of 0.001 M in DMSO. The obtained data indicate the formation of the target complexes in the form [Co(AA)(H2O)2]NO3.2H2O, [Ni(AA)(H2O)2]Cl.4H2O, [Cu(AA)]Cl.2.5H2O, [Eu(AA)(H2O)Cl]Cl.6H2O and [Gd(AA)(H2O)(NO3)]NO3.2H2O. Co and Ni gave Oh structure while Cu gave square planar form. The Crystal field parameters calculated and discussed for the target complexes. Molecular modelling, molecular mechanics and DFT calculation carried out to the synthesized compounds. The x-ray analysis indicates the nanoparticle behaviour for the Cu-AA complex with an orthorhombic structure. The interactions of the synthesized complexes with DNA moiety investigated with means of spectrometric titration (UV–vis. spectra) and by using fluorescence spectroscopy. Antimicrobial activities of the synthesized compounds screened using the disc diffusion method. HAA and Cu-AA gave activity exceeded the Ampicillin. The docking work carried using the targeting protein of Escherichia coli FabH (PDB code: 1HNJ). The obtained binding energy compared and discussed in terms of the in vitro studies.