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Aims: The production of peptaibols, toxic secondary metabolites of Trichoderma, in the indoor environment is not well‐documented. Here, we investigated the toxicity of peptaibols in the guttation droplets and biomass of Trichoderma strains isolated from problematic buildings. Methods and Results: Seven indoor‐isolated strains of T. atroviride, T. trixiae, T. paraviridescens and T. citrinoviride were cultivated on malt extract agar, gypsum boards and paperboards. Their biomass extracts and guttation droplets were highly cytotoxic in resting and motile boar sperm cell assays and in inhibition of somatic cell proliferation assays. The toxins were identified with HPLC/ESI‐MS/MS as trichorzianines, trilongins, trichostrigocins and trichostrigocin‐like peptaibols. They exhibited toxicity profiles similar to the reference peptaibols alamethicin, trilongins, and trichorzianine TA IIIc purified from T. atroviride H1/226. Particular Trichoderma strains emitted the same peptaibols in both their biomasses and exudate droplets. The trilongin‐producing T. citrinoviride SJ40 strain grew at 37°C. Conclusions: To our knowledge, this is the first report of indoor‐isolated Trichoderma strains producing toxic peptaibols in their guttation droplets. Significance and Impact of the Study: This report proves that indoor isolates of Trichoderma release peptaibols in their guttation droplets. The presence of toxins in these types of exudates may serve as a mechanism of aerosol formation for nonvolatile toxins in the indoor air. [ABSTRACT FROM AUTHOR]