Volatile spiroketals are well-documented semiochemicals secreted by beetles and wasps for the intra- and interspecies communication. Its use in insect traps and as natural herbicide makes them of commercial interest. Besides insects, fungi are well-known producers, but the fungal biogenesis of spiroketals has remained speculative. Product formation along fatty acid degradation based on non-labeled feeding experiments was assumed. Thus, the observed occurrence of conophthorin and (E)- and (Z)-chalcograns in submerged cultures of the basidiomycete Trametes hirsuta prompted a precursor study aiming at a more detailed insight into their formation. Supplementation of (9Z,12 Z)-octadecadienoic (linoleic) acid resulted in elevated product yields and the identification of a fourth spiroketal, 2,8-dimethyl-1,7-dioxaspiro[5.5]-undecane. However, no intermediates of fatty acid degradation suitable as spiroketal precursors were identified. In addition, the hyphae lacked lipoxygenase activity, which was formerly supposed to be mandatory for spiroketal formation. Supplementation of 1-/2-13C acetate showed incorporation of the label into chalcogran. Therefore, a formation along the polyketide pathway analogous to insects was concluded. Graphical abstract: [Figure not available: see fulltext.]