It is commonly known that the pretreatment of complex substrates yields higher biogas production in anaerobic digestion (AD) by improving hydrolysis. However, it is still questioned whether all solubilized fractions after pretreatment can be used for CH4 production during AD. In this study, the relationship between increased solubilization and AD efficiency in response to different pretreatment conditions of lipid-extracted microalgae waste (LEMW) was investigated. The individual pretreatment (acid and ultrasonic) and combined pretreatment were applied to assess the solubilization of LEMW. A biochemical methane potential (BMP) test was subsequently performed to determine the AD efficiency. Combined pretreatment of LEMW (60 min of irradiation + pH 1) showed the highest performance, achieving CH4 production of 1245 ± 28 mL CH4/L with increased solubilization of 50.4%. However, it was found that increased solubilization did not proportionally increase CH4 productivity. The assessment of the origin of produced CH4 through biomass fractionation supports this finding in that the soluble fraction that does not contribute to CH4 production increased at more severe pretreatment conditions.