Materials formulated La2Ni1−xCuxO4+δ (0 ≤ x ≤ 1) have been prepared by the nitrate-citrate route, having an orthorhombic K2NiF4 structure with Fmmm (x = 0) and Bmab (0.1 ≤ x ≤ 1) space groups. La2NiO4+δ, which resulted to be the compound with the highest capability to accommodate excess oxygen (δ = 0.16), underwent heat treatments (873–1098 K) under high oxygen pressure (200–250 bar). This led to an increase of excess oxygen in the structure. Rietveld refinements on neutron powder diffraction data probe the excess of oxygen to be accommodated as interstitial defect at the atomic position (1/4, 1/4, z; z ≈ 1/4), which requires the displacement of four neighbouring oxygen atoms from their normal positions. This result is in agreement with a non-stoichiometry model which induces the stabilization of oxygen vacancies in the perovskite layer of these materials. The defect concentration determined from Rietveld refinement agrees well with the data obtained from thermogravimetric analysis. Results obtained herein reveal heat treatment under high oxygen pressure (873 K, 200 bar) as a promising method to enhance transport properties in K2NiF4-type structures.