New lipopeptide homologues (AF, AF, and AF) with antifungal activities against and spp. were purified from a cell-free supernatant of RLID 12.1. The lipopeptides AF, AF, and AF were identified with the same peptide sequence Asn-Pro-Tyr-Asn-Gln-Thr-Ser with variations in the fatty acid branching type and chain length (-C, -C, and -C, respectively). Upon comparing the three homologues for MICs against 81 ( = 64) and ( = 17) clinical isolates and their cytotoxicities, we found that AF was the most promising antifungal lipopeptide, since it demonstrated 100% inhibition at geometric mean MICs of 3.31, 3.41, 3.48, and 2.83 μg/ml against ,, , and, respectively, with low hemolysis values (<6%) and 50% inhibitory concentrations (13.31 μg/ml). The additive effects among the homologues AF, AF, and AF were evaluated against three species, along with the cytotoxicity studies. Five combinations exhibited good additive interaction effects: AF/AF (at corresponding concentrations of 4 and 4 μg/ml [4/4 μg/ml]), AF/AF (4/4 μg/ml), AF/AF (2/4 μg/ml), AF/AF (4/4 μg/ml), and AF/AF (2/4 μg/ml) in planktonic cell inhibition and AF/AF (4/4 μg/ml), AF/AF (4/4 μg/ml), and AF/AF (2/4 μg/ml) in the inhibition of biofilm formation. However, combinations AF/AF and AF/AF, which showed >70% cell survival with low hemolysis (<5%), were found to be comparatively effective. We describe here the additive effects of lipopeptide homologues showing reduced cytotoxicity against mammalian cells; these combinations might serve as a potent antibiofilm-forming substitute.