<p>A novel ammonium trans-1,2-cyclohexanediaminetetraacetato copper(II) dihydrate complex, NH₄[Cu(Hcdta)]·2H₂O, was synthesized and thoroughly characterized by elemental analysis, FT-IR, electronic spectroscopy, thermal analysis, and single-crystal X-ray diffraction. The complex features a five-coordinate distorted square pyramidal geometry around the Cu(II) center, coordinated by two nitrogen and three oxygen atoms, with one carboxymethyl group uncoordinated. Its structure is further stabilized by extensive hydrogen bonding network involving coordinated and lattice water molecules, confirmed through Hirshfeld surface analysis. Spectroscopic data revealed characteristic d–d and ligand-to-metal charge transfer transitions, while thermal studies showed stepwise dehydration, ammonia loss, and final decomposition to Cu₂O. Biological investigations demonstrated strong intercalative binding with calf thymus DNA, supported by UV–Vis spectral hypochromism, red shifts, and fluorescence enhancement. The complex exhibited moderate antibacterial and potent antifungal activity comparable to standard drugs, alongside selective cytotoxicity against human lung (A549), breast (MCF-7), and cervical (HeLa) cancer cell lines, with minimal toxicity toward normal keratinocytes. These results highlight NH₄[Cu(Hcdta)]·2H₂O as a promising multifunctional agent for antibacterial and anticancer applications, highlighting the impact of the cyclohexane backbone in CDTA on metal coordination and biological function.</p>