Fructooligosaccharides (FOS) are short fructans with different degrees of polymerization (DP) and bonds in their structure, generated by the distinct activities of fructosyltransferase enzymes, which produce distinct types of links. FOS are in high demand on the market, mainly because of their prebiotic effects. In recent years, depending on the link type in the FOS structure, prebiotic activity has been shown to be increased. Studies on β-fructanofuranosidases (Ffasa), enzymes with fructosyltransferase activity in yeasts, have reported the production of F-FOS, F-FOS, and G-FOS. The aims of this investigation were to evaluate the capability of fifteen different yeasts to grow in sp. juices and to determine the potential of these juices as substrates for FOS production. Additionally, the research aimed to corroborate and analyze the fructosyltransferase activity of enzymatic extracts obtained from agave yeasts by distinct induction media and to identify the role and optimal parameters (time and sucrose and glucose concentrations) for FOS and disaccharides production through Box-Behnken designs. To carry out such a task, different techniques were employed: FT-IR, TLC, and HPAEC-PAD. We found two yeasts with fructosyltransferase activity, ITMLB97 and ITMLB85. In addition, within the most relevant results, the production of the FOS 1-kestose, 6-kestose, and neokestose, as well as disaccharides inulobiose, levanobiose, and blastose, molecules with potential applications, was determined. Overall, FOS production requires suitable yeast species, which grow in a medium under optimal conditions, from which microbial enzymes with industrial potential can be obtained.