AbstractIn preceding studies the neotropical ascomycete Hypoxylon rickii turned out to be a prolific source of new secondary metabolites, considering that we had obtained terpenoids with five different scaffolds along with a series of terphenyls. From the mycelial extracts of a 70 L scale fermentation of this strain we additionally isolated nine new macrolides (1–9) by RP‐HPLC. The planar structures were elucidated by NMR spectroscopy complemented by HR‐ESIMS. The relative configurations were assigned by J‐based configuration analyses and confirmed by Kishi′s Universal Database. Subsequently, the absolute configurations were assigned by Mosher′s method using the shift analysis of a tetra‐MTPA derivative. For rickiol A (1) and E (5) we observed transesterification of 20‐membered ring structures to 22‐membered isomers rickiol A2 (6) and E2 (7), and to 24‐membered isomers rickiol A3 (8) and rickiol E3 (9), respectively. Cytotoxic effects and moderate antibiotic activity against Gram‐positive bacteria were observed for 1–8 and 1–6 and 8, respectively. The total synthesis of rickiol E3 (9) established easier access to these compounds.