A two-stage temperature-phased mesophilic anaerobic digestion assay was carried out to study the interaction between various biological pretreatment conditions and the possible synergistic co-digestion of microalgae and primary sludge. The study of growth kinetics of the biochemical methane potential test revealed that a maximum of 36% increase in methane yield was observed from co-digestion of a substrate pretreated by thermophilic aerobic conditions (55 °C and HRT = 2 days) and an 8.3% increase was obtained from the anaerobic pretreated substrate (55 °C and HRT = 3 days). Moreover, no synergistic effects on methane yields were observed in co-digesting the substrate pretreated with high temperature (85 °C). The study also identified specific conditions in which interaction between biological pretreatment and co-digestion might substantially reduce methane yield. Careful optimization of operating conditions, both aerobic and anaerobic pretreatment at moderate thermophilic conditions, can be used as a biological pretreatment to enhance methane yield from the co-digestion of microalgae and primary sludge.