This study presents a rapid and comprehensive method for screening mushroom extracts for the putative discovery of bioactive molecules, including those exhibiting antimicrobial activity. This approach utilizes a panel of bioluminescent bacteria, whose light production is a sensitive indicator of various cellular effects triggered by the extracts, including disruption of bacterial communication (quorum sensing), protein and DNA damage, fatty acid metabolism alterations, and oxidative stress induction. The bioassay’s strength is its ability to efficiently analyze a large number of extracts simultaneously while also assessing several different mechanisms of toxicity, significantly reducing screening time. All samples analyzed exhibited more than one cellular effect, as indicated by the reporter bacteria. Four samples (C. cornucopioides, F. fomentarius, I. obliquus, and M. giganteus) displayed the highest number (six) of possible mechanisms of antibacterial activity. Additionally, combining extraction and purification protocols with a bioluminescent bacterial panel enables simultaneous improvement of the desired antimicrobial properties of the extracts. The presented approach offers a valuable tool for uncovering the diverse antimicrobial mechanisms of mushroom extracts.

This study evaluated the biomedical potential of ethanolic extracts from five native Thai mushrooms, revealing distinct biological activities associated with their metabolomic profiles. Termitomyces clypeatus R. Heim demonstrated exceptional biocompatibility, effectively promoting human keratinocyte (HaCaT) proliferation without causing cytotoxicity to the cells. Its primary metabolome, rich in essential amino acids and nucleosides, supports its potential as a candidate for topical wound healing, pending further functional validation. Conversely, Lentinus squarrosulus exhibited strong antibiofilm properties and moderate direct antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa, demonstrating a weak additive effect with gentamicin. Mechanistic bioreporter screening and Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS) suggested that this efficacy may be associated with quorum-sensing disruption and the presence of bioactive hydroquinolones, in addition to potential genotoxic stress induction. While Schizophyllum commune displayed moderate effects, Volvariella volvacea and Thaeogyroporus porentosus exhibited high-dose cytotoxicity linked to severe oxidative stress and high organic acid content, limiting their direct high-dose biomedical applications. Ultimately, T. clypeatus R. Heim and L. squarrosulus have emerged as promising candidates that warrant further investigation for tissue regeneration and potential adjunctive anti-infective applications.