Green roofs are increasing in popularity in the urban environment for their contribution to green infrastructure; but their role for biodiversity is not often a design priority. Maximising biodiversity will impact positively on ecosystem services and is therefore fundamental for achieving the greatest benefits from green roofs. Extensive green roofs are lightweight systems generally constructed with a specialised growing medium that tends to be biologically limited and as such can be a harsh habitat for plants to thrive in. Thus, this investigation aimed to enhance the soil functioning with inoculations of soil microbes to increase plant diversity, improve vegetation health/performance and maximise access to soil nutrients. Manipulations included the addition of mycorrhizal fungi and a microbial mixture (‘compost tea’) to green roof rootzones, composed mainly of crushed brick or crushed concrete. The study revealed that growing media type and depth play a vital role in the microbial ecology of green roofs, with complex relationships between depth and type of substrate and the type of microbial inoculant applied, with no clear pattern being observed. For bait plant measurements (heights, leaf numbers, root/shoot biomass, leaf nutrients), a compost tea may have positive effects on plant performance when grown in substrates of shallower depths (5.5 cm), even one year after inoculums are applied. Results from the species richness surveys show that diversity was significantly increased with the application of an AM fungal treatment and that overall, results suggest that brick-based substrate blends are most effective for vegetation performance as are deeper depths (although this varied with time). Microbial inoculations of green roof habitats appeared to be sustainable; they need only be done once for benefits to still been seen in subsequent years where treatments are added independently (not in combination). They seem to be a novel and viable method of enhancing rooftop conditions.