In recent years the relationship between the intestinal microbiota, the host and chronic non-communicable diseases has brought interest into the study of its formation and maintenance in the host. Lactic acid bacteria (BAL) are Gram-positive bacteria with probiotic activity, which have been associated with many health benefits, such as decreased body fat mass and lower risk of type II diabetes mellitus. One of the main colonization mechanisms and bacteria survival strategies is the production of biofilms and the use of prebiotics as substrates to achieve a balance within intestinal microbiota. However, there is not enough evidence to demonstrate the biofilm formation in the presence of agave fructans (AF). This study aimed to evaluate in vitro the biofilm formation in a consortium of lactic acid bacteria: Lactobacillus delbrueckii ssp. lactis, Lactobacillus delbrueckii ssp. bulgaricus y Streptococcus thermophilus in the presence of AF at different concentrations: 0%, 0,1%, 4%, 8% y 16%. The addition of 0,1% of AF correlates with the best capacity for biofilm formation. The findings imply the possibility of modulating the biofilm formation of lactic acid bacteria with AF. These results can contribute positively to the host, by generating intestinal homeostasis, colonization resistance, stability to food digestion and chemical modifications of drugs and carry out beneficial functions to the health.