Cholera, caused by the Gram-negative bacterium Vibrio cholerae, remains a significant world-wide public health issue, causing severe and often fatal diarrhoeal illness in humans. V. cholerae biofilm has been suggested to play a critical role in the pathogenesis and disease transmission. However, the role of intestinal glycans in biofilm formation during infection, as well as their role in adhesion of V. cholerae to host cells, colonization and virulence, remains poorly understood. In this study we explored the involvement of Vibrio polysaccharide (VPS), Lipopolysaccharide (LPS) and biofilm matrix proteins in the presence of monosaccharides. Glycan array analysis of multiple V. cholerae strains revealed that common glycans interacted with bacterial cells, such as gangliosides (GM1), blood group antigens and Lewis antigens for both the wild type strain and its rugose variant, a VPS overexpressing variant of the wild type. The VPS, LPS and biofilm matrix protein mutants all had greatly reduced ability to interact with these glycans. Interestingly, only the rugose variant and VPS mutant strains showed the ability to bind to blood group H and P antigens, while biofilm matrix protein and LPS mutants lost these bindings, indicating that an overproduction of VPS may allow for additional interactions with the host cell. Some of the important glycans that had the ability to interact with the V. cholerae cells were galactose, mannose, glucosamine, fucose, and in addition, mucin. Biofilm assays showed that galactose had an inhibitory effect on the biofilm formation by the rugose variant and the LPS mutant, while it had a stimulatory effect on the biofilm formation of the biofilm matrix protein mutant. Mannose was revealed to have an inhibitory effect on the biofilm formation of the VPS and LPS mutants, while a stimulatory effect was noticed on the biofilm formation of the biofilm matrix protein mutants. Glucosamine had an inhibitory effect on the biofilm formation by the wild type strain and a stimulatory effect on the biofilm formation of the biofilm matrix protein mutant. Fucose and mucin were shown to have an inhibitory effect on the biofilm formation by the wild type strain, its rugose variant, the VPS, LPS and biofilm matrix protein mutants. In addition, the level of adhesion displayed by the wild type, its rugose variant, and its VPS, LPS and biofilm matrix protein mutant strains to Caco-2 cells were investigated. The results revealed severe adhesion deficiencies in the VPS, LPS and biofilm matrix protein mutant strains. Environmental and clinical strains were also tested for adhesion with Caco-2 cells but yielded no discernible pattern regarding adhesion capabilities. One environmental strain and one clinical strain displayed severe adhesion deficiencies, while the other environmental and clinical strain displayed a greater adhesion than the wild type. Biofilm assays revealed that, when compared to the biofilm formation of the smooth wild type strain, all environmental and clinical strains tested had an increase in biofilm formation. The study and testing of more environmental and clinical strains may reveal an association in biofilm formation between environmental and disease settings.