Applying green manure and plant growth-promoting fungi (PGPF) can contribute to sustainable agricultural production, reduce the use of chemical fertilisers, and preserve soil biodiversity. The available research literature does not extensively cover the agricultural advantages associated with the simultaneous utilisation of green manure along with PGPF as a substitute for chemical fertilisers. In particular, little attention has been paid to the effects of combining green manure with PGPF on soil enzyme activities, fungal biomass and population, and plant growth promotion. Therefore, we conducted to clarify the effects of the combined application of green manure and the Trichoderma spp. strain RW309 having phosphorus- and carbon-related enzyme activities, plant growth-promoting potential, and also influence on soil fungal community. Two green manure treatments [Vicia villosa (hairy vetch) and Brassica juncea (L.)] with and without RW309, only RW309, and control (only soil), were applied. Soil samples were collected after the co-application of treatments and after plant harvesting soil. This is the first finding that plant growth was significantly higher with green manure combined with RW309 inoculation than with green manure alone. The enzyme activities and fungal biomass were also markedly higher in the co-application treatments than in the other treatments. It is likely that the co-incorporation of green manure and RW309 decomposes organic matter faster and provides an energy source to soil microbes. Moreover, new finding was to enhance specific fungal genera (Penicillium spp.), which may be responsible for nutrient mineralisation and plant growth promotion. In conclusion, co-application of green manure and beneficial microbial strains should be promoted for sustainable agricultural practices to improve soil health and maintain sustainable agriculture.