Utilization of crude glycerol (CG) from the biodiesel industry in the production of biogas offers a perspective of further energy generation, which may result into the drop of biodiesel costs on the developing world market. This contribution is focused on anaerobic treatment of CG as a single substrate in mixed laboratory reactors. Experiences from long-term operation of mixed reactors processing either untreated or acidulated CG are discussed. The possibility of cofermentation of washing water (WW) from biodiesel production with CG was also attempted. It was demonstrated that long-term mesophilic anaerobic treatment of CG as the only substrate is possible. Except for nitrogen, and possibly phosphorus, the addition of other nutrients is unnecessary. Processing of both non-acidulated and acidulated CG in laboratory mixed reactors inoculated with suspended sludge resulted in a stable operation with high specific methane production (0.328 L/g chemical oxygen demand (COD) for non-acidulated CG and 0.345 L/g COD for acidulated CG), regarding organic loading rate of up to 4 g COD/(L x d). Due to the considerable content of dissolved inorganic salts in CG it is recommended to dilute this substrate with water to prevent the accumulation of salts and inhibition of the biomass activity. WW was proved to be a problematic substrate for anaerobic cofermentation with CG because its addition to the reactor caused a decrease in the pH value and biogas production.