For the production of bioethanol production from lignocellulosic biomass, pretreatment is an important step for breaking the chemical bond present in lignocelluloses. Among the different pretreatment strategies, biological pretreatment is found to be a promising approach and is eco-friendly and produces less inhibitors. In this process, lignolytic microorganisms, such as bacteria and fungi, are used to reduce the recalcitrance of feedstock and finally enhance the digestibility with biomass hydrolyzing enzymes. Microbial-based pretreatment is usually carried out by growing microbes directly into the biomass or using enzyme cocktails. Challenges of biological pretreatment in pilot-scale studies include a longer incubation time when compared to conventional pretreatment strategies. This challenge can be overcome by utilization of potent microbial consortium. This chapter presents an overview of the merits and limitations of biological pretreatment, factors affecting biological pretreatment, as well as future perspectives.