Nanomaterials, which promote the generation of reactive oxygen species (ROS) and precipitate the onset of oxidative stress responsible for the development of a variety of adverse conditions, have a great prospect for various nanomedicine applications. In this paper, for the first time, we report the pro-oxidant activity of GdYVO₄:Eu³⁺ and GdVO₄:Eu³⁺ nanoparticles (NPs) in water solutions without any external stimuli (in the darkness). This dark pro-oxidant activity is shown to be associated with superoxide (O₂•–) and hydroxyl radical (•OH) generation and is dependent on the size of NPs and, consequently, the concentration of surface defects, such as oxygen vacancies VO. For small 2 nm GdYVO₄:Eu³⁺ NPs, strong O₂•– and •OH radical production has been revealed, whereas for larger GdVO₄:Eu³⁺ NPs, low level of O₂•– and negligible level of •OH radical production have been detected during the sample storage in the darkness. The possible mechanisms of the dark ROS generation in ReVO₄:Eu³⁺ NPs are considered.