Background Aspergillus niger mycelium waste, a by-product from citric acid fermentation is considered a good raw material to produce fungal chitosan and glucosamine (GlcN). However, its successful application has always been a challenge.Results In this work, the effects of combined pretreatment involving steam explosion and ionic liquid (IL) on mycelium waste were investigated. Results show that pretreatment of mycelium waste at 2.5 MPa for 1 min improved the GlcN hydrochloride yield by 65.49%, the release of soluble sugar reached 0.46 g/g of sample, and the deacelation degree of fungal chitosan by hydrolysis of chitin deacetylase from Rhodococcus equi CGMCC14861 (ReCDA) was from scratch and a 1.3-fold increase by combined with IL pretreatment. In addition, a recycling strategy for by-products, which promoted the fermentation of ReCDA used for fungal chitosan, was efficiently developed.Conclusion This is the first report of comprehensive utilization of Aspergillus niger mycelium waste from citric acid fermentation on the basis of steam explosion and ionic liquid pretreatment. This study demonstrates a green-like technology for the comprehensive utilization of A. niger mycelium waste from citric acid fermentation.