Green roofs provide ecosystem services through evapotranspiration and nutrient cycling that depend, among others, on plant species, substrate type, and substrate depth. However, no study has assessed thoroughly how interactions between these factors alter ecosystem functions and multifunctionality of green roofs. We simulated some green roof conditions in a pot experiment. We planted 20 plant species from 10 gen-era and five families (Asteraceae, Caryophyllaceae, Crassulaceae, Fabaceae, and Poaceae) on two substrate types (natural vs. artificial) and two substrate depths (10 cm vs. 30 cm). As indicators of major ecosystem functions, we measured aboveground and belowground biomasses, foliar nitrogen and carbon content, foliar transpiration, substrate water retention, and dissolved organic carbon and nitrates in leachates. Interactions between substrate type and depth strongly affected ecosystem functions. Biomass production was increased in the artificial substrate and deeper substrates, as was water retention in most cases. In contrast, dissolved organic carbon leaching was higher in the artificial substrates. Except for the Fabaceae species, nitrate leaching was reduced in deep, natural soils. The highest transpiration rates were associated with natural soils. All functions were modulated by plant families or species. Plant effects differed according to the observed function and the type and depth of the substrate. Fabaceae species grown on natural soils had the most noticeable patterns, allowing high biomass production and high water retention but also high nitrate leaching from deep pots. No single combination of factors enhanced simultaneously all studied ecosystem functions, highlighting that soil–plant interactions induce trade-offs between ecosystem functions. Substrate type and depth interactions are major drivers for green roof multifunctionality.