This work reports an improved process of enzymatic saccharification on Agave salmiana leaves and its conversion into bioethanol comparing strains of Saccharomyces cerevisiae ethanol RED and Kluyveromyces marxianus OFF1. A. salmiana leaves were pretreated with acid-alkaline sequential process before the application of several treatments of enzymatic saccharification. In the results, the best enzymatic treatment reached conversion of 94.49%, releasing sugar concentrations of 50 g L-1. During fermentation, glucose consumption efficiencies were similar for both strains with values of 98%, while the consumption rates were higher for S. cerevisiae. Fermentative efficiencies were higher for K. marxianus with values of 92.88 +/- 3.24% while for S. cerevisiae they were 87.63 +/- 2.23%. These findings show a saccharification enzymatic process efficiently applied on A. salmiana leaves. K. marxianus, a non-Saccharomyces strain, converted all the sugars released from this plant structure into bioethanol with similar values or even higher than a Saccharomyces strain. (C) 2019 Elsevier Ltd. All rights reserved.