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 °C), durations (15, 30 ± 15 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 °C, 700 °C, and 900 °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 (Ms) values from 23.53 to 55.15 emu/g. Chromium substitution showed the best antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans. 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.