The new Schiff base ligand N,N′-thiophene-(bis-1-ethyl-6,8-difluoro-7-(3-methylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3carboxylic acid) (H2L) was synthesized via condensation of thiophene-2,5-diamine with lomefloxacin in ethanol using glacial acetic acid. H2L reacted as tetradentate ligand with chromium(III), manganese(II), and cobalt(II). The synthesized compounds were characterized using elemental analysis, molar conductivity (Λm), magnetic susceptibility (µeff), FTIR, UV–Vis, GC–MS, and thermal studies were employed to ensure the chelation process. Infrared measurements confirmed that H2L chelated with metal ions via the carboxylate oxygen and nitrogen of the azomethine group also, with water molecules. UV–Vis and magnetic moment data supported octahedral geometries for all complexes. The calculated optical band gap energy (Eg) values suggested that our complexes were more electro-conductive relative to H2L. Thermal analyses revealed the presence of significant lattice and coordinated water molecules within the complexes’ coordination spheres. Thermodynamic parameters including activation energy, entropy change, enthalpy change, and Gibbs free energy change were derived using Coats–Redfern and Horowitz–Metzger methods. Disc diffusion method was implemented for assessing antimicrobial effects versus some pathogenic microorganisms. The data revealed that Cr(III) complex as most effective against Escherichia coli and Staphylococcus aureus. Cytotoxicity tests on normal prostate epithelial cells (IC50 = 6.30–10.43 µM) indicated significant toxicity and limited selectivity, restricting anticancer applicability.