In the present work, a new ligand N'1, N'2-bis((E)-2-hydroxybenzylidene) oxalohydrazide (H4OAS) and its chelates, [Mn2(H2OAS)(H2O)2Cl2].H2O and [Fe2(OAS)(H2O)4Cl2]0·5H2O, have been synthesized and elucidated through a variety of physicochemical techniques, including elemental analysis, melting point, 1H and 13C-NMR, electronic spectra, Fourier-transform infrared spectroscopy (FT-IR), and thermogravimetric analysis (TGA). Utilizing the Gaussian 9 software and density functional theory (DFT), computations were done to evaluate the geometries for the synthesized ligand and its complexes. Magnetic studies and electronic spectra suggested that Fe(III) chelate has an octahedron coordination structure, while Mn(II) chelate has a tetrahedral coordination structure. In addition, the IR spectral data showed that the ligand exhibits a binegative tetradentate behavior in [Mn2(H2OAS)(H2O)2Cl2].H2O and as a tetranegative hexadentate in [Fe2(OAS)(H2O)4Cl2]0·5H2O. The thermal study of the prepared complexes has been done and led us to determine the activation parameters of several decomposition steps. The kinetic parameters of the resulting thermal decomposition steps were calculated using two methods (Coats-Redfern and Horowitz-Metzger). Based on the calculated activation energy, the thermal stability order was found to be [Fe2(OAS)(H2O)4Cl2]0·5H2O>[Mn2(H2OAS)(H2O)2Cl2].H2O. The ligand and complexes have also been screened for their antibacterial and antifungal potency against multi-stranded microorganisms including gram-negative bacteria (Escherichia coli), gram-positive bacteria (Staphylococcus aureus), and fungal species (Candida albicans). Furthermore, the in-vitro cytotoxic activity of the prepared compounds has been tested against human hepatocellular carcinoma cells (HePG-2) and the results showed that H4OAS and its Fe(III) complex have average activity against HePG-2 with IC50 values of 9.2 and 11.3 µmol/L, respectively. Finally, the synthesized ligand was utilized as a chelating agent and oleic acid as a surfactant in the ion-flotation procedure to separate Fe(III) from the aqueous media.