The reaction of 6-amino pencillanic acid with Schiff base “(Z)-3-((3-hydroxybenzylidene) amino) phenol)" has formed a novel Azo–azomethine ligand (HL) “(7-((Z)-(2-hydroxy-4-((Z)-((3-hydroxyphenyl) imino) methyl) phenyl) diazenyl)-3, 3- dimethyl-6-oxo-2-thiabicyclo [3.2.0] heptane-4-carboxylic acid". The ligand (HL) is characterized by 1H-NMR, 13 C-NMR, Mass Spectrometry, CHNs, Uv-Visible, and FT-IR. This new Ligand (HL) is used to prepare numerous complexes using transition metal ions Fe (II) Cu (II), and Ni (II). These complexes' properties are studied using different techniques such as Mass Spectroscopy, Atomic Absorption, FT-IR, UV-Visible, Magnetic Moment, and Molars Conductivity. All these techniques indicate 1:2 (M: L) stoichiometry's synthesized complexes. Azo-azomethine ligand (HL) acts as a tridentate ligand to form the octahedral shape complexes with sp3d2, d2sp3 Hybridization. These new ligands and complexes are studied for their biological activity. The results show that the minimum inhibition concentration (MIC) of the prepared compounds has inhibition activity against Staphylococcus aureus bacteria and obviously affects the ability of pathogenic bacteria (S. aureus) to attain virulence factors such as Biofilm and hemolysin toxin