<jats:title>Abstract</jats:title><jats:p>Five new polyketides that contain tetramic acids, myceliothermophins A–E, were isolated from the thermophilic fungus <jats:italic>Myceliophthora thermophila</jats:italic>. Two sets of 5‐alkyl‐5‐hydroxyl (or 5‐methoxyl)‐1<jats:italic>H</jats:italic>‐pyrrol‐2(5<jats:italic>H</jats:italic>)‐one diastereomers, myceliothermophins A/B and C/D, were separated as pure compounds by using silica‐gel column chromatography and recycling reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The relative configurations of the chiral centers in 5‐alkyl‐5‐hydroxyl (or 5‐methoxyl)‐1<jats:italic>H</jats:italic>‐pyrrol‐2(5<jats:italic>H</jats:italic>)‐one moieties were deduced from NOESY correlations. In the cytotoxic assay, the 5‐(2‐methylpropyldiene)‐1<jats:italic>H</jats:italic>‐pyrrol‐2(5<jats:italic>H</jats:italic>)‐one analogue (myceliothermophin E) exhibited inhibition against four cancer cell lines. In addition, the significant inhibitory effect of myceliothermophins A and C and the inactivity of myceliothermophins B and D revealed the importance of the relative configurations of 5‐alkyl‐5‐hydroxyl (or 5‐methoxyl)‐1<jats:italic>H</jats:italic>‐pyrrol‐2(5<jats:italic>H</jats:italic>)‐one moieties on their cytotoxicity potency against cancer cells.