<p>A novel henna-green/microwave-assisted hydrothermal method was applied to synthesize cobalt-manganese spinel ferrite nanocomposites. This eco-friendly approach, utilizing henna extract as a capping agent, combined with a microwave-assisted hydrothermal technique, offers advantages such as environmental sustainability, time, and energy efficiency. Examined parameters included pH (10 ± 1), temperatures (175 ± 25&#xa0;°C), durations (15, 30 ± 15&#xa0;min), and forty milliliters of henna extract prepared with varying henna quantities (five, ten, or fifteen grams per hundred milliliters of water). Post-optimization, various substituents like nickel and chromium were explored. Samples were calcined at 500&#xa0;°C, 700&#xa0;°C, and 900&#xa0;°C, and analyzed using Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and magnetic property assessments. XRD revealed a cubic face-centered spinel ferrite and hematite structure. SEM and TEM showed diverse morphologies (rod-like, flower-like, polygonal) influenced by synthesis conditions and substituents. XPS confirmed Co²⁺, Mn²⁺, Ni²⁺, Fe³⁺, and Cr³⁺ presence in the spinel lattice. The nanocomposites exhibited ferromagnetic behavior with saturation magnetization (M_s) values from 23.53 to 55.15&#xa0;emu/g. Chromium substitution showed the best antimicrobial activity against <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, and <i>Candida albicans</i>. This study highlights the potential of the henna-green/microwave-assisted hydrothermal method for controlled synthesis of substituted cobalt spinel ferrite nanocomposites with tunable properties for various applications.</p>