Abstract In the context of biofuel production and achieving sustainable bioeconomy, the use of lignocellulosic and algae biomass in anaerobic fermentation processes yields biohythane that has a typical composition of 10–15% H2, 50–55% CH4 and 30–40% CO2. Using organic biomass-based substrates has been shown to minimize environmental impacts due to the versatile production of high-value products under normal operating conditions that are practically achievable. However, the biohythane yield depends on different factors such as the biomass type, the organic loading rate, soluble metabolic products formed, the type of fermentation (single/dual stage) and the pretreatment strategy adopted for the biomass. Different pretreatment strategies based on physical, chemical and biological processes have been proposed in the literature. In this review, improvements in biohythane yield as a result of these pretreatment strategies, the need/effect of inoculum enrichment, the effects of pH, temperature, trace element addition and organic loading rate has been reviewed. Finally, the major developments of improving biohythane yield due to the addition of co-substrates and the current trends are discussed.