The herbaceous perennial energy crops miscanthus, giant reed, and switchgrass, along with the annual crop residue corn stover, were evaluated for their bioconversion potential. A co-hydrolysis process, which applied dilute acid pretreatment, directly followed by enzymatic saccharification without detoxification and liquid–solid separation between these two steps was implemented to convert lignocellulose into monomeric sugars (glucose and xylose). A factorial experiment in a randomized block design was employed to optimize the co-hydrolysis process. Under the optimal reaction conditions, corn stover exhibited the greatest total sugar yield (glucose + xylose) at 0.545 g g−1 dry biomass at 83.3% of the theoretical yield, followed by switch grass (0.44 g g−1 dry biomass, 65.8% of theoretical yield), giant reed (0.355 g g−1 dry biomass, 64.7% of theoretical yield), and miscanthus (0.349 g g−1 dry biomass, 58.1% of theoretical yield). The influence of combined severity factor on the susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernible, showing that co-hydrolysis is a technically feasible approach to release sugars from lignocellulosic biomass. The oleaginous fungus Mortierella isabellina was selected and applied to the co-hydrolysate mediums to accumulate fungal lipids due to its capability of utilizing both C5 and C6 sugars. Fungal cultivations grown on the co-hydrolysates exhibited comparable cell mass and lipid production to the synthetic medium with pure glucose and xylose. These results elucidated that combining fungal fermentation and co-hydrolysis to accumulate lipids could have the potential to enhance the utilization efficiency of lignocellulosic biomass for advanced biofuels production. Biotechnol. Bioeng. 2013; 110: 1039–1049. © 2012 Wiley Periodicals, Inc.