The methane yield from the digestion of algae is typically much lower than the theoretical methane yield, and lower than yields reported for other organic substrates. This study presents a novel free nitrous acid (FNA) pre-treatment technique to improve methane production from algal biomass. The methane production yield through anaerobic digestion was found to be dramatically enhanced by FNA pre-treatment (2.31 mg HNO2–N L−1), with a 51% increase in the methane yield (from 161 to 250 L CH4 per kg VS added). A two substrate model was used to describe the apparent presence of rapid and slowly degradable material. Model-based analysis revealed that with FNA pre-treatment (2.31 mg HNO2–N L−1), the availability of both rapid and slowly biodegradable substrates were increased. Higher levels of nitrite (159 and 1006 mg N L−1) had an inhibitory/toxic effect. For this reason, coupled with the fact that denitrification of nitrite consumes organic substrate, it is concluded that pre-treatment liquor should be removed before digestion.