Biofilm formation plays a key role in the life cycles and subsistence of many microorganisms. For the human fungal pathogen Candida albicans, biofilm development is arguably a virulence trait, because medical implants that serve as biofilm substrates are significant risk factors for infection. The development of C. albicans biofilms in vitro proceeds through an early phase, in which yeast cells populate a substrate, an intermediate phase, in which pseudohyphal and hyphal cell types are produced, and a maturation phase, in which continued cell growth is accompanied by accumulation of an extracellular matrix. Here we report the results of a screen for C. albicans biofilm-defective mutants, in which homozygous insertions in NUP85, MDS3, KEM1, and SUV3 were found to block biofilm development. Confocal microscopic examination suggests that nup85, suv3, and mds3 mutations cause early-phase arrest, whereas the kem1 mutation causes intermediate-phase arrest. All of the mutants are defective in hypha production in several media. Analysis of mixed-biofilm development indicates that all of the mutants are defective in the production of hyphae in the context of a biofilm. Because all of the mutants are defective in the retention of cells in the biofilm, we infer that hyphae provide an adherent scaffold that stabilizes the biofilm structure.