Abstract Alkaline and biological pretreatments were compared for enhancing the biological methane potential of Napier grass. The earlier reported biotreatments for Napier grass did not use the edible white-rot fungus Pleurotus sajor-caju, as in the present work. Dry Napier grass was ground to different particles sizes (20–30 mm, N1-L; ≤0.6 mm, N1-S). The N1-L grass was treated with alkali and designated as the alkali treated grass N2. The samples N1-S, N1-L and N2 were used separately as substrates for growing the fungus for 14 days at room temperature (30 ± 2 °C) in a solid-state biotreatment. Alkali treatment delignified the grass 2.1- to 10.7-fold better than the fungus. Fungal treatment resulted in 3.8- to 8.3-fold loss in glucan compared to alkali treatment. Maximum xylan loss occurred in the N1-S fine-ground grass after fungal growth. The fungus-grown grass samples (N1-FL, N1-FS, N2-F), the untreated ground samples (N1-L, N1-S) and the alkali treated sample (N2) were anaerobically digested to determine the biological methane potential. The fungus-grown grass samples had a maximum daily methane production in the range of 44–50 cm3 g VS−1, significantly higher than the samples not treated with the fungus. The alkali treated grass gave a significantly higher cumulative methane yield than the untreated grass and the biological methane potential was ∼71–77% of the theoretical methane potential. The proportion of methane in the total gas produced from the treated grass was in range of 74–83% by volume whereas it was 57–68% for the untreated grass.