Bark, an underutilized biomass resource, is a promising raw material for cellulose nanofibrils (CNF) production due to its renewability, abundance, and low cost. Because data on the physical and chemical properties of bark-based CNF is scarce, we investigated the feasibility of CNF production from two different bark species (Norway spruce and silver poplar) using a combination of sequential extraction, delignification, and mechanical fibrillation. The chemical composition, crystallinity index, morphology, fibril diameter, thermal stability, and mechanical properties of the CNF were evaluated and compared with those of wood-based CNF from the same species. The results showed that spruce bark yielded CNF virtually undistinguishable from spruce wood-derived CNF in terms of structure and mechanical properties of nanopapers made thereof. Nanopapers prepared from spruce wood and bark showed comparable tensile strength and Young's modulus of around 130 MPa and 15 GPa, respectively. Contrarily, CNF derived from poplar bark were different in structure compared to poplar wood CNF, and nanopapers produced from poplar bark CNF showed only modest mechanical performance. In summary, the extensive characterization carried out demonstrated the general feasibility of CNF production from tree bark but also revealed the need for further optimization, like in the case of poplar bark.