Volatile terpenes (VTs) content in tree-ring resin, in response to natural infection by Heterobasidion spp. in asymptomatic mature Norway spruce (Picea abies) trees was investigated. Twenty-three randomly selected mature trees were sampled in a stand in the Western Italian Alps by extracting cores using an increment borer. Based on fungal isolations from cores and molecular typing using taxon-specific competitive-priming (TSCP)-polymerase chain reaction, 12 out of the 23 trees were identified as infected by Heterobasidion parviporum. Tree-ring growth patterns and VT content in tree rings were determined. Analysis of VT content was performed by means of gas chromatography mass spectrometry on a subset of trees. Results show slightly but not significantly lower tree-ring width in infected compared with non-infected trees in the past two decades. Total concentrations of sesquiterpenes (SQTs) and relative proportions of α-pinene, β-pinene and longifolene were significantly greater in infected trees; while relative proportions of camphene, 3-carene, ρ-cymene, sesquiterpene 15.90 and α-farnesene were significantly lower. This is the first study showing that VTs in tree-ring resin may indicate infection of trees by a fungal forest pathogen, even when trees are mostly asymptomatic.