Abstract Cation mixing is a well-recognized mean to obtain oxides of desired functionality with predetermined structure and stoichiometry, which yet has been only little analyzed at the nanoscale. In this context, we present a comparative analysis of the stability and mixing properties of O-poor and O-rich two-dimensional V-Fe oxides grown on Pt(111) and Ru(0001) surfaces, with the aim of gaining an insight into the role of substrate and oxygen conditions on the accessible Fe contents. We find that due to the high oxygen affinity of the Ru substrate, the mixed O-rich layers are highly stable while the stability of O-poor layers is limited to inaccessibly oxygen-poor environments. In contrast, on the Pt surface, O-poor and O-rich layers coexist with, however, a much lower Fe content in the O-rich phase. We show that cationic mixing (formation of mixed V-Fe pairs) is favored in all considered systems. It results from local cation-cation interactions, reinforced by a site effect in O-rich layers on the Ru substrate. In O-rich layers on Pt, Fe-Fe repulsion is so large that it precludes the possibility of substantial Fe content. These findings highlight the subtle interplay between structural effects, oxygen chemical potential, and substrate characteristics (work function and affinity towards oxygen), which governs the mixing of complex 2D oxide phases on metallic substrates.