Ageratina adenophora originating from central America has flooded forests, pastures, and farmland in more than 40 tropical and subtropical countries, causing huge ecological disasters and economic losses. In this paper, we intended to use a complex inoculum composed of Pseudomonas putita and Clostridium thermocellum to in-situ compost A. adenophora debris and then to compare the phytotoxicity of extracts from uncomposted and composted A. adenophora (UCA and CA respectively) to barley seed germination and young seedling growth. A field experiment was finally conducted to reveal the effects of UCA and CA on barley nutrients uptake, yield, grain quality, soil enzyme activities, microbial biomass and biodiversity. In-situ composting sharply decreased 4,7-dimethyl-1-(propan-2-ylidene)-1,4,4a,8a-tetrahydronaphthalene- 2,6(1H,7H)-dione(DTD) and 6-hydroxy-5-isopropyl-3,8-dimethyl-4a,5,6,7,8,8a-hexahydronaphthal en-2(1 H)-one(HHO) from 2096.3 and 743.7 mg kg-1 in uncomposted A. adenophora to 194.4 and 68.19 mg kg-1 in composted A. adenophora. UCAE showed negative influences on seed germination performances (except lower rates on germination percentage). The mechanism may be the inhibition of bio-macromolecules hydrolysis (including proteins, starch, and phytin) in endosperms and their hydrolysates for forming new plants. CAE promoted seed germination and seedling growth, increased chlorophyll levels in leaves, and stimulated dehydrogenase and nitrate reductase activities in plants, while UCAE got opposite performance. Compared with chemical fertilizers, application of CA in combination with chemical fertilizers significantly improved plant nutrient uptake (nitrogen, phosphorus, and potassium), yield, grain quality, quantity of 16S rDNA sequences, richness and diversity of bacterial communities in contrast to UCA which behaved otherwise. Taken together, the use of the microbial agent to in-situ compost A. adenophora may be an effective approach for agricultural use of A. adenophora debris as a plant-friendly organic fertilizer, being undoubtedly worth advocating.