Laccase detoxification has been proposed to enhance the enzymatic hydrolysis of lignocellulosics by reducing the effect of inhibitors formed during the pretreatment step. Herein, we investigated the effect of the laccase treatment on the enzymatic hydrolysis of two lignocellulosic materials pretreated by diluted acid/steam explosion through two strategies. Simultaneous laccase treatment and enzymatic hydrolysis of pretreated corn stover (PCS) and sugar cane straw (PSCS) led to a significant reduction of the concentration of glucose released from 67.7 down to 39.2 g/Kg and from 69 down to 53.5 g/Kg. Laccase treatment prior to hydrolysis of both substrates showed the same inhibitory effect. After hydrolysis of PCS and PSCS, the glucose concentration decreased by approximately 44 and 21% compared to the control assays, respectively. Therefore, these results do not support the use of laccases to detoxify pretreated lignocellulosic materials for improving the bioethanol production.