This study performs natural sand-based synthesis using the sonochemical route for preparing Zn-doped magnetite nanoparticles. The nanoparticles were dispersed in water as a carrier liquid to form Zn-doped magnetite aqueous ferrofluids. Structural data analysis indicated that the Zn-doped magnetite nanoparticles formed a nanosized spinel structure. With an increase in the Zn content, the lattice parameters of the Zn-doped magnetite nanoparticles tended to increase because Zn2+ has a larger ionic radius than those of Fe3+ and Fe2+. The existence of Zn-O and Fe-O functional groups in tetrahedral and octahedral sites were observed in the wavenumber range of 400-700 cm-1. The primary particles of the Zn-doped magnetite ferrofluids tended to construct chain-like structures with fractal dimensions of 1.2-1.9. The gas-like compression (GMC) plays as a better model than the Langevin theory to fit the saturation magnetization of the ferrofluids. The ferrofluids exhibited a superparamagnetic character, with their magnetization was contributed by aggregation. The Zn-doped magnetite ferrofluids exhibited excellent antibacterial activity against gram-positive and negative bacteria. It is suggested that the presence of the negatively charged surface and the nanoparticle size may contribute to the high antibacterial activity of Zn-doped magnetite ferrofluids and making them potentially suitable for advanced biomedical.