Basidiomycete fungus Serpula lacrymans is one of the most dangerous indoor fungus causing dry rot of timber. The physiology of this fungus deserves more attention as a basis for development of methods of dry rot treatment. We observed an increase in the freezing resistance of S. lacrymans after pre-cultivation of mycelia at elevated temperatures. To examine the biochemical mechanisms underlying this phenomenon the lipid composition and metabolite profiling of mycelia subjected to freezing and thawing were investigated. An analysis is made of the growth rate and metabolism of “daughter” cultures derived from a frozen mycelia. According to the results, sphingolipids and water-soluble metabolites such as mannitol, glycerol, sugar alcohols, some amino- and organic acids are able to function as protective compounds providing a cross-resistance between heat shock and freeze–thaw stress in S. lacrymans.