Abstract The potential of microbial pretreatment of cotton stalks by Phanerochaete chrysosporium to degrade lignin and facilitate fuel ethanol production was investigated under two culture conditions: submerged cultivation (SmC) and solid state (SSC) cultivation. Although microbial pretreatments showed significant lignin degradation (LD) (19.38% and 35.53% for SmC and SSC, respectively), a study on hydrolysis and fermentation of the microbial-pretreated cotton stalks showed no increase in cellulose conversion (10.98% and 3.04% for SmC and SSC pretreated samples, respectively) compared to untreated cotton stalks (17.93%). Solid state cultivation demonstrated better selectivity of 0.82 than 0.70 with submerged pretreatment. Washing of pretreated cotton stalks did not significantly increase cellulose conversion. However, heating and washing remarkably improved ( P<0.05) cellulose conversion to 14.94% and 17.81% for SmC and SSC 14 day pretreatment, respectively. Ethanol yields, up to 0.027 g ethanol g −1 initial cotton stalks, were low for all untreated and pretreated samples mainly due to the low cellulose conversion. Although potential and some critical aspects of fungal pretreatment using P. chrysosporium have been explored in this study, additional investigation is still required especially to improve the selectivity for preferential LD and to optimize hydrolysis efficiency. The mechanism of catalytic binding of cellulolytic enzymes to cotton stalks as affected by the presence of fungal mycelia also warrants further study.