Variability in bioactivity and chemical composition of Eucalyptus bosistoana F. Muell. heartwood extracts between individual trees from two different sites were investigated. Combining the results of fungal assays and quantitative gas chromatography (GC) of the extracts allowed the investigation of bioactive compounds. The bioactivity of extracts was assessed against white rot (Trametes versicolor [L.] Lloyd) and brown rot (Coniophora cerebella Pers.). Ethanol extracts from E. bosistoana heartwood were less effective on the white rot than against the brown rot. Variability in the bioactivity of extracts against the two fungi was observed between the trees. A site effect in the bioactivity was found for the white rot but not the brown rot. Bioactivity of the extracts against the white rot was not correlated to that against the brown rot. The absence of a relationship between of effects of the extracts on the relative growth rates of the white rot and the brown rot indicated that the two fungi were affected by different compounds. Thirty two compounds were quantified in E. bosistoana ethanol extracts by GC, of which six (benzoic acid, hexadecanoic acid, 1,5-dihdroxy-12-methoxy-3,3-dimethyl-3,4-dihydro-1H-anthra[2, 3-c]pyran-6,11-dione, octadecanoic acid, polyphenol and beta-sitosterol) were identified. Significant variability in eight compounds was found between the two sites. Multivariate (PLSR) analysis identified compounds at the retention times 10.2 and 11.5 min (hexadecanoic acid) to be most related to the bioactivity of the E. bosistoana heartwood extracts against white rot and brown rot.