The aim of this work was to find out if biofilms can be made by lactic acid bacteria (LAB) isolated from agave plants using agave fructans as sole carbohydrate substrates or if it was necessary to use fructose as a breakdown product of such polymers. This is part of a research project geared to develop industrial lactic acid production from agave fructans, an abundant raw material in Mexico’s agave plantations. Present results showed that nine strains of LAB isolated from Agave salmiana and belonging to genus Lacticaseibacillus and Enterococcus produced exopolysaccharides directly from agave fructans to a greater extent than with fructose. The best polysaccharide productions in planktonic cultures were Lacticaseibacillus paracasei strains DG2, DG3, DG4 and DG8. Furthermore, all nine LAB strains produced biofilms on polystyrene microplates, much better with agave fructans than with fructose. In most strains, biofilm formation was favored at pH from 6.0 to 6.5, except for strains DG7 and DG9 where pH 5.5 was optimal. Biofilm formation required between 3 and 5 days of incubation in all Lacticaseibacillus paracasei strains, whereas Enterococcus faecium required a little less of 3 days. Present results support the straight use of agave fructans to develop LAB biofilms using agave epiphytic bacteria. This finding simplifies upstream processing of agave fructans to be used for future lactic acid fermentation in LAB biofilm reactors.