Dysbiosis of the gut microbiome has been implicated in inflammatory bowel diseases. We have shown that levels ofCandida tropicalis, along with those ofEscherichia coliandSerratia marcescens, are significantly elevated in Crohn’s disease (CD) patients. Here, we evaluated the ability of a novel probiotic to prevent and treat polymicrobial biofilms (PMB) formed byC. tropicaliswithE. coliandS. marcescens. SinceCandida albicanshas been reported to be elevated in CD patients, we investigated the interactions ofC. albicanswith these bacterial species in biofilm formation. We determined whether the interaction betweenCandidaspp. and bacteria is specific by usingTrichosporon inkinandSaccharomyces fibuligeraas comparators. Additionally, the effects of probiotics onC. albicansgermination and biofilm formation were determined. To determine the ability of the probiotic to prevent or treat mature biofilms, probiotic filtrate was added to the PMB at early (prevention) and mature (treatment) phases. Biofilm thickness and architecture were assessed by confocal scanning laser microscopy. The effects of the probiotic on germination were evaluated in the presence of serum. Exposure ofC. tropicalisPMB to probiotic filtrate reduced biofilm matrix, decreased thickness, and inhibited hyphal formation. We showed thatC. albicansorC. tropicalisformed significantly thicker PMB than control biofilms, indicating that this interaction isCandidaspecific. Treatment with probiotic filtrate inhibitedC. albicansgermination and prevented/treatedC. albicansPMB. The designed probiotic may have utility in the management of biofilm-associated gastrointestinal diseases such as Crohn’s and colorectal cancer.IMPORTANCEThe effects of diversity of the gut microbiome on inflammation have centered mainly on bacterial flora. Recent research has implicated fungal species and their interactions with other organisms in the inflammatory process. New ways to restore microbial balance in the gut are being explored. Our goal was to identify beneficial probiotic strains that would antagonize these fungal and bacterial pathogens that are elevated in the inflamed gut, and which also have antibiofilm activity. Fungus-bacterium correlation analysis allowed us to identify candidate probiotic species that can antagonize microbial pathogens, which we subsequently incorporated into a novel probiotic formulation. Amylase, which is known to have some antibiofilm activity, was also added to the probiotic mixture. This novel probiotic may have utility for the management of inflammatory bowel diseases by disrupting polymicrobial biofilm formation.