House dust mites are major causative agents in the pathogenesis of allergy. Their proximity with human habitats, association with development of allergenic diseases, and resistance to physical and chemical control measures; make them some of the most medically important mites. Understanding of house dust mites has been hampered by a lack of genomic sequence data and limited to a discrete number of proteins. The work presented here is a detailed characterisation of the European house dust mite, Dermatophagoides pteronyssinus airmid strain, at the genomic and proteomic level. Genomic sequencing and assembly resulted in a high-quality assembly of 70.76 Mb in size with 96.86% coverage. A comprehensive bioinformatic and proteomic examination was conducted on the 12,530 predicted proteins, validating the expression of 4,002. A small group of D. pteronyssinus airmid proteins showed significant homology to known allergens from other species. A large scale comparative proteomic investigation of the mite body and spent growth medium allowed for: (i) qualitative assessment of allergen localisation and (ii) the identification of numerous enzymes that may be involved in key physiological activities. The characterisation of protein extracts from house dust also identified a substantial number of uncharacterised D. pteronyssinus proteins in addition to known and putative allergens. The genes encoding novel β-1,3 glucanases were identified within a trigene cluster in D. pteronyssinus airmid. Recombinant protein expression, biochemical and proteomic analysis revealed Glu1 and Glu2 to exhibit hydrolytic activity toward β-1,3 glucans and have increased expression in the mite body and excretome of D. pteronyssinus in response to yeast diet. Further proteomic and enzymatic analysis correlated glucanase activity in house dust with presence of Glu1 and Glu2. These findings provide evidence that active β-1,3 glucanases are expressed and excreted in the faeces of D. pteronyssinus in response to fungal diet, in both the laboratory and the wild-type environment.