Two new isochromans named pseudoanguillosporin A (2a) and B (3), together with the known cephalochromin (1), were isolated from Pseudoanguillospora sp. The C‐2 absolute configuration of 2a and 3 was deduced from its CD spectrum by the isochroman helicity rule, supported by TDDFT CD calculations. The absolute configuration of the sec‐hydroxyl group of 3 was determined by the Mosher NMR method from the MPA esters of its methylated derivative. The axial chirality of 1 was assigned through exciton analysis of its CD spectrum and confirmed by ZINDO CD calculations. The metabolites showed broad antimicrobial activities.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)