This study investigated the mycochemical composition and bioactive potential of three wild mushroom species—Cortinarius trivialis, Mycena pura, and M. rosea—sampled from Tara Mountain, Serbia. The focus was on their polyamine content, total phenolics, total flavonoids, antioxidant capacity (DPPH, ABTS, NO, and FRAP assays), and acetylcholinesterase (AChE) inhibitory activity. Polyamines (PAs) (putrescine, spermidine, spermine) were quantified in lyophilized fungal samples using high-performance liquid chromatography with fluorescence detection (HPLC-FD). In addition, total phenolics, total flavonoids, antioxidant capacity, and anti-AChE activity were determined from 80% methanolic extracts using previously established spectrophotometric methods. Quantification of putrescine (PUT), spermidine (SPD), and spermine (SPM) revealed C. trivialis as particularly enriched in PUT (47.51 ± 15.92 mg/kg d.w.) and SPD (41.58 ± 18.06 mg/kg d.w.), while M. pura and M. rosea contained lower amounts. M. pura stood out for its high phenolic and flavonoid content, driving strong antioxidant activity across DPPH, ABTS, FRAP, and NO radical assays. M. rosea displayed selective ABTS scavenging and the strongest AChE inhibition (93.24 ± 7.90%), whereas C. trivialis, despite modest antioxidant activity, combined rich polyamine levels with notable AChE inhibition. Principal component analysis underscored distinct metabolic signatures: phenolic-driven antioxidant defense in M. pura, SPD-associated neuroprotection in M. rosea, and high PUT accumulation in C. trivialis.

To the best of our knowledge, this is the first report linking polyamine profiling with AChE inhibition in these mushroom species. The findings highlight a species-specific interplay of polyphenols and polyamines in shaping antioxidant and neuroprotective activities, positioning these inedible fungi as promising reservoirs for bioactive compounds with potential applications in functional foods, nutraceuticals, and neuroprotective strategies.