This study describes the preparation of a new bidentate Schiff base derived from the condensation of Isatin-3-hydrazone with 2-acetylthiophene and the preparation of new series of complexes with a good yield. The prepared ligand was characterized by IR, UV-Vis, C.H.N.S elemental analysis, 1H and 13C NMR, LC-Mass spectroscopy, and physical measurements. Its complexes were analyzed by C.H.N.S elemental analyses, UV-Vis., FTIR, NMR, LC-Mass Spectra, atomic absorption spectroscopy, magnetic susceptibility, and conductivity measurements The results from spectroscopy and measurement studies showed that the ligand coordinated to the metal ion as a bidentate ligand via oxygen and nitrogen, forming an octahedral geometry around it. In vitro antimicrobial activity of the prepared compounds was tested against Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Bacillus bacteria strains, and Candida fungi by the agar well diffusion method. The ligand and its complexes have a good effect on the studied microorganisms' Gram-positive and Gram-negative strains and fungi.