A new group of Cu(II) and Fe(III) complexes has been produced utilizing the ligand (Z)-N′-(1-(thiophen-2-yl)ethylidene)-2-(p-tolylamino)acetohydrazide (H2L). The chemical structure of new compounds has been established using FT-IR, 1H NMR, electronic spectra, powder X-ray diffraction, and thermal behavior. The Gaussian 09 program used the Density Functional Theory (DFT) approach to optimize the geometry of all synthesized compounds to acquire the best possible structures and significant parameters. The examination of biological aspects included the study of microbial and breast cancer cell lines. It was found that both the ligand and the complexes exhibited strong antibacterial activity against the studied bacterial species. The complexes worked as strong antifungal agents filamentous fungi like Candida albicans. The H2L ligand and its Cu(II) complexes (M1 and M2) were more effective against Candida albicans than Fe(III) complex, which showed no activity against Aspergillus niger. Both mononuclear and binuclear copper (II) complexes expanded their antifungal capabilities to target Aspergillus niger, surpassing the effectiveness of the standard drug. Amphotericin was used as a reference. Following testing against the MCF-7 breast cancer cell line, the reactivity of all compounds was investigated. All complexes showed an intriguingly powerful antiproliferative potential. The compounds’ activities were organized like this: Cu(II) Complexes (M2, M1) > Fe(III) Complex (M3) > H2L Ligand. Molecular docking simulation identified the active amino acids relevant to microbial and breast cancer studies.