The metabolites of the mycobiont culture of the lichen Trypethelium eluteriae were isolated by column chromatography and preparative TLC. Nine compounds (1–9) including two new trypethelones, 8-methoxytrypethelone (6) and 5′-hydroxy-8-ethoxytrypethelone (9), together with four known trypethelones (3–4, 7–8), and two known phenalenones (1–2) were characterized. It is the first report of 8-methoxytrypethelone methyl ether (5) purification as a racemic mixture in T. eluteriae. Earlier,

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... -methoxyltrypethelone (10) was reported as new compound with erroneous spectroscopic data. This compound was identified later as 8-hydroxytrypethelone methyl ether (4). X-ray crystallographic structures of compounds 5–7 were elucidated for the first time. Phenalenones (1–2) and trypethelones (5–6 and 9) were the additional compounds discovered in the cultured mycobiont of T. eluteriae. Six compounds (1–2, 5–8) were screened against Mycobacterium tuberculosis H37Rv and two compounds (7–8) against non-tuberculosis mycobacteria and other human pathogenic bacteria. Compound (7) inhibited M. tuberculosis H37Rv strain with an MIC of 12.5 µg/mL.