The hydroxylase activities of new strains such as Curvularia lunata, C. geniculata, C. eragrostidis, C. prasadii, Ulocladium botrytis, Alternaria tenuis, and Fusarium oxysporum toward three steroid substrates, namely, androstenedione (AD), cortexolone (S), and dehydroepiandrosterone acetate (DAA), were characterized. The 9α-hydroxylase activity of C. lunata 1011 cells against S to form 9α-hydroxy-S was shown for the first time. It was found that C. geniculata 837 and F. oxysporum 11dn1 strains can hydroxylate substrates to form pharmacologically promising 7α-hydroxysteroids. C. geniculata 837 cells selectively hydroxylate AD, resulting in 7α-hydroxytestosterone, whereas F. oxysporum 11dn1 leads to the transformation of DAA to 7α-hydroxydehydroepiandrosterone.