Aureobasidins (Ab's) are antifungal antibiotics produced by Aureobasidium pullulans R106, with over 20 congeners (e.g., AbA) structured as cyclic depsipeptides containing eight amino acids (three or four N-methylated) and one hydroxy acid. We discovered six new congeners (AbT1-T4, AbU1-U2) and report their isolation, structures, and antifungal activities. The fermentation broth was treated with ethanol, then purified using HP-40 resin, silica-gel column, and ODS-silica HPLC. Structures were determined via amino acid analysis, high-resolution fast atom bombardment mass spectrometry (HRFAB-MS), and stereochemical analysis (chiral columns), revealing specific substitutions relative to AbA: AbT1 contains (2R,3S)-2-hydroxy-3-methylpentanoic acid [(2R,3S)-Hmp] at position 1; AbT2 has N-methylleucine (MeLeu) at position 7; AbT3 has D-2-hydroxyisovaleric acid (D-Hiv) at position 1, β-hydroxy-N-methylphenylalanine (βHOMePhe) at position 4, and N-methylvaline (MeVal) at position 9; AbT4 has N-methylalloisoleucine (Mealle) at position 7; AbU1 has valine (Val) at position 2; AbU2 has D-Hiv at position 1 and Val at position 2. Antifungal activity assays (minimum inhibitory concentration, MIC) showed AbT1 had higher activity against Candida albicans than AbA (MIC 0.0125 vs. 0.05 μg/ml), AbU1 had the lowest activity (MIC 0.78 μg/ml), and AbT3 was the most active among congeners lacking β-hydroxy-N-methylvaline (βHOMeVal) at position 9. N-methyl groups at positions 2 and 7 are critical for the high antifungal activity of Ab's, while the role of βHOMePhe in AbT3 contradicted previous findings with AbR (which showed little activity despite having βHOMePhe and MeVal).