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The preparation of Co-doped magnetite ferrofluids from natural sand was developed using a double-layer technique. The Co-doped magnetite nanoparticles formed a spinel phase with lattice parameters in the range of 8.355-8.422 Å and tended to agglomerate with the particle sizes of 7-12 nm. The presence of the first and second layers from oleic acid and DMSO was detected by the infrared spectrum as well as the olive oil used as a carrier liquid. The saturation magnetization of the superparamagnetic samples decreased from 24.4 to 4.8 emu/g with decreasing Co 2+ composition. The particle size and electrostatic forces between the magnetic particles and the microbes played an essential role in inhibiting microbial growth. Interestingly, the increasing Co 2+ composition enhanced the superior performance of the ferrofluids against E. coli, S. aureus, B. subtilis, and C. albicans . With additional extensive investigation, we believe that the prepared Co-doped magnetite double-layered ferrofluids from natural sand with superior antimicrobial performance can be new significant antimicrobial agents., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.., (© 2020 The Author(s).)