This paper presents the synthesis and characterisation of a triple-phase magnetic nanostructured composite - CuO/Fe2O3/CuFe2O4. The CuO/Fe2O3/CuFe2O4 was evaluated for antimicrobial efficacy and underlying mechanisms, as well as potential ecotoxicity using the marine bacterium Vibrio fischeri and freshwater microcrustacean Daphnia magna. Human safety was assessed in vitro using an immortalized keratinocyte cell line. Antimicrobial activity was tested against Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa, Gram-positive bacteria Staphylococcus aureus, and the fungus Candida albicans at concentrations ranging from 1 to 1000 mg/L in deionized water. After 4 h, the minimum bactericidal concentration (MBC) of CuO/Fe2O3/CuFe2O4 was 250 mg/L for E. coli and 500 mg/L for P. aeruginosa. Prolonged exposure for 24 h reduced the MBC to 10 mg/L for and 100 mg/L, respectively. S. aureus was less sensitive, showing MBC of 100 mg/L only after 24 h, while the minimum fungicidal concentration towards C. albicans was 1000 mg/L at 4 h and 500 mg/L at 24 h. The studies indicated that the antimicrobial effects resulted from the combined action of generated reactive oxygen species, shed metal ions, and particle-cell interactions due to composite’s positive surface charge. No acute ecotoxicity for V. fischeri, and low toxicity effects for D. magna were observed. CuO/Fe₂O₃/CuFe₂O₄ also showed no cytotoxicity to human keratinocytes in vitro at concentrations up to 500 mg/L upon 24 h exposure. Overall, CuO/Fe₂O₃/CuFe₂O₄ exhibited uniform magnetism, antimicrobial activity, and initial indications of low (eco)cytotoxicity, making it a promising candidate for applications that require a balance between antimicrobial efficacy and safety.