β-Peptides (β-amino acid oligomers) that mimic the amphiphilic, helical, and cationic properties of natural antimicrobial peptides have previously been shown to display antifungal activity against planktonic Candida albicans cells. β-Peptides offer several advantages over conventional peptides composed of α-amino acid residues, including conformational stability, resistance to proteases, and activity at physiological salt concentrations. We examined sequence-activity relationships toward both planktonic C. albicans cells and C. albicans bio-films, and the results suggest a toxicity mechanism involving membrane disruption. A strategy for fluorescently labeling a β-peptide without diminishing antifungal activity was devised; labeled β-peptides penetrated the cell membrane and accumulated in the cytoplasm of both planktonic and biofilm-associated cells. The labeled β-peptide was detected only in metabolically inactive cells, which suggests that β-peptide entry is correlated with cell death. The presence of a β-peptide at a concentration near the minimum inhibitory concentration completely prevented planktonic C. albicans cells from forming a biofilm, suggesting that β-peptides may be useful in preventing fungal colonization and biofilm formation.