This study investigated the role of liquid nitrogen (LN2) in increasing microbial accessibility of wool proteins for biogas production. It involves a mechanical size reduction of four different types of raw wool fibres, namely, Blackface, Bluefaced Leicester, Texel and Scotch Mule, in presence of liquid nitrogen, followed by the determination of the methane production potential of the pre-treated wool fibres. The highest methane yield, 157.3 cm3 g−1 VS, was obtained from pre-treated Scotch mule wool fibre culture, and represented more than 80% increase when compared to the yield obtained from its raw equivalent culture. The increase in biogas yield was attributed to the effectiveness of LN2 in enhancing particle size reduction and the consequent increase in wool solubility and bioavailability. Results also showed that LN2 pre-treatment can enhance size reduction but has limited effect on the molecular structure. The study also showed that the biogas potential of waste wool fibres varies with the type and source of wool.