<p>Structural and quantitative changes in lipid and osmolyte profiles can serve as markers of technogenic stress caused by heavy metal pollution. This study investigates the biochemical responses of common soil filamentous fungi (<i>Alternaria septospora</i>, <i>Cladosporium halotolerans</i>, <i>Fusarium equiseti</i>, <i>Trichoderma harzianum</i>, and <i>Clonostachys farinosa)</i> to copper (Cu) exposure, focusing on changes in lipids (membrane and storage lipids) and specific osmolytes (polyols and certain carbohydrates). Based on effective concentration values, <i>A. septospora</i> and <i>C. farinosa</i> proved to be the most Cu-resistant species. Under Cu stress, we observed an increased phosphatidylcholines/phosphatidylethanolamines (PC/PE) ratio in the melanized <i>A. septospora</i>, <i>C. halotolerans</i>, and the resistant <i>C. farinosa</i>. Conversely, Cu exposure led to an increased proportion of phosphatidic acids in <i>T. harzianum</i>. Changes in osmolyte composition included elevated mannitol levels, alongside reduced levels of low molecular weight polyols (arabitol, erythritol) and carbohydrates, primarily trehalose. The increased PC/PE ratio, elevated mannitol, and reduced low molecular weight polyols may serve as reliable indicators of Cu-induced stress. These findings underscore the pivotal role of lipid and osmolyte remodeling in fungal tolerance to copper stress and suggest their potential utility as biochemical markers for assessing environmental heavy metal contamination and guiding bioremediation strategies.</p>