Previously we described the isolation and structural elucidation of bergofungin (1) using mass spectrometry (FAB; B/E-MIKES (E)-scan; ESI-CID-MS/MS) and NMR spectroscopic methods, which belongs to the peptaibols characterized by a high portion of helix-promoting α-aminoisobutyric acid (Aib) in their linear peptide backbone molecules, containing an amino alcohol at the carbon terminus and acetylated nitrogen terminus, and displaying membrane-modifying properties with various bioactivities. A more detailed study of the producing strain Emericellopsis donezkii HKI 0059 unraveled the presence of three additional homologous bergofungins B (2), C (3), and D (4). In this paper, we report their isolation and structures. The producer strain was cultivated as surface culture in malt medium, harvested, and extracted with ethyl acetate. The extract was purified via silica gel chromatography and isocratic preparative HPLC. Compared with bergofungin A (1), 2 and 3 are distinguished by different amino acid moieties in positions 8 and 12, while 4 has a 14-membered amino acid backbone. Their molecular weights were determined by HRFAB-mass spectrometry, with physicochemical properties including appearance, ESI-MS, melting point, IR, and HPLC retention time summarized. Compounds 2, 3 and 4 display antimicrobial activity against Sporobolomyces salmonicolor SBUG549 and Bacillus subtilis ATCC6633 at concentrations >50 µg/ml, and along with bergofungin A (1), moderately inhibit the activity of prolyl endopeptidase from Flavobacter sp. with Ki 0.18 to 0.34 µM.