Novel methods for species detection based on collection of environmental DNA (eDNA) are not only important in biodiversity assessment in a scientific context, but are also increasingly being applied in conservation practice. The eDNA-based biodiversity detection methods have significant potential for regular use in biodiversity status assessments and conservation actions in protected areas (PAs) and other effective area-based conservation measures (OECMs) worldwide. Species detection based on DNA from environmental samples, such as water, sediment, soil, air, or organic material, has a broad application scope with precise, comprehensive, and rapid species identification. Here, we provide an overview of the application range of eDNA-based methods for biodiversity monitoring in PAs, evaluate environmental assessments in which this technology has already been implemented for nature conservation, and examine the challenges that can hamper further application in real world practice. Based on the outcomes of two projects, practical experience, and current scientific literature focusing on their application, we conclude that eDNA-based species detection methods provide promising novel approaches that have strong potential as supplement methods, or in some cases even as substitutes for the conventional monitoring methods used for PAs. This advancement is expected to affect decision-making in biodiversity conservation efforts in PAs and OECMs.