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Purpose: Contact lenses have direct contact with the corneal surface and can induce sight‐threatening infection of the cornea known as Acanthamoeba keratitis. The objective of this study was to evaluate the dysprosium‐based nanoparticles (Dy‐based NPs), namely Fe3O4‐PEG‐Dy2O3 nanocomposites and Dy(OH)3 nanorods, as an active component against Acanthamoeba sp., as well as the possibility of their loading onto contact lenses as the drug administering vehicle to treat Acanthamoeba keratitis (AK). Methods: The Dy‐based NPs were synthesized, and they were loaded onto commercial contact lenses. The loading content of the NPs and their release kinetics was determined based on the absorbance of their colloidal solution before and after soaking the contact lenses. The cytotoxicity of the NPs was evaluated, and the IC50 values of their antiamoebic activity against Acanthamoeba sp. were determined by MTT colorimetric assay, followed by observation on the morphological changes by using light microscopy. The mechanism of action of the Dy‐based NPs against Acanthamoeba sp. was evaluated by DNA laddering assays. Results: The loading efficiencies of the Dy‐based NPs onto the contact lens were in the range of 30.6–36.1% with respect to their initial concentration (0.5 mg ml–1). The Dy NPs were released with the flux approximately 5.5–11 μg cm–2 hr–1, and the release was completed within 10 hr. The emission of the NPs consistently showed a peak at 575 nm due to Dy3+ ion, offering the possible monitoring and tracking of the NPs. The SEM images indicated the NPs are aggregated on the surface of the contact lenses. The DNA ladder assay suggested that the cells underwent DNA fragmentation, and the cell death was due most probably to necrosis, rather than apoptosis. The cytotoxicity assay of Acanthamoeba sp. suggested that Fe3O4‐PEG, Fe3O4‐PEG‐Dy2O3, Dy(NO3)3.6H2O and Dy(OH)3 NPs have an antiamoebic activity with the IC50 value being 4.5, 5.0, 9.5 and 22.5 μg ml–1, respectively. Conclusions: Overall findings in this study suggested that the Dy‐based NPs can be considered as active antiamoebic agents and possess the potential as drugs against Acanthamoeba sp. The NPs could be loaded onto the contact lenses; thus, they can be potentially utilized to treat Acanthamoeba keratitis (AK). [ABSTRACT FROM AUTHOR]