Formation of fungal biofilms on health care-related materials causes serious clinical consequences. This study reports a novel fungal repelling strategy to control fungal biofilm formation on denture biomaterials through layer-by-layer self-assembly (LBL). Amphiphilic quaternary ammonium chitosans (CS612) were synthesized and used as the antimicrobial positive layer, and sodium alginate (SA) was chosen as the negative layer to construct LBL multilayers on poly (methyl methacrylate) (PMMA)-based denture materials. The presence of LBL multilayers on denture disc was confirmed and characterized by surface zeta potential, water contact angle and FT-IR analyses. The multilayer coatings, especially CS612 as the outmost layer, effectively prevented the fungal initial adhesion and biofilm formation. The Candida cells avoided the multilayer coatings and suspended in broth solution instead of forming biofilms, suggesting that the LBL multilayers had fungal repelling effects. The LBL multilayers were biocompatible toward mammalian cells. In stability tests, after immersion in PBS for 4 weeks under constant shaking and repeated brushing with a denture brush for up to 3,000 times, the biofilm-controlling effects of the LBL multilayers were not affected, pointing to a novel long-term strategy in controlling fungal biofilms on denture and other related biomaterials.