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Ex vivo interaction of NaYF 4 :Yb,Er nanophosphors with isolated mitochondria has been investigated. The nanophosphors were synthesized using the hydrothermal method. The synthesized NaYF 4 :Yb,Er nanophosphors were characterized for physicochemical properties. The NaYF 4 :Yb,Er nanophosphors showed successful upconversion with excitation wavelength lying in the nearinfrared region. The effect of synthesized NaYF 4 :Yb,Er nanophosphors on mitochondria isolated from the chicken heart tissue was examined through ROS generation capacity, membrane fluidity, and complex II activity. The exposer of NaYF 4 :Yb,Er nanophosphors to isolated mitochondria inhibits ROS generation activity as compared to control. The mitochondria membrane fluidity of the lipid bilayer and complex-II activity of mitochondria was observed to be unaltered after the interaction with NaYF 4 :Yb,Er nanoparticles. The results confirm that synthesized NaYF 4 :Yb,Er nanoparticles can be used as a safe contrast agent.