Anaerobic co-digestion provides a promising solution for converting inexpensive carbon from wastes to biogenic methane. We used microalgae (Nannochloropsis oculata) with cow manure and sludge to produce a better quantity and quality of biogas. To further improve the gas production, microalgae were pretreated with ultrasonication, hot water, and a combination of both. Interestingly, the results showed that the pretreatment of microalgae decreased biogas production by 5 to 30%. The no-pretreatment runs produced a maximum of 118 L of biogas. The relative content of biogenic methane was higher in the pretreated feedstock (48 to 52%) in comparison with the no-pretreatment runs (44%). The conversion of volatile suspended solids present in the feedstock to total biogenic methane production was highest in hot-water-treated runs. The carbon content in the gas produced by the pretreated microalgae peaked (38%) in the middle of the experiment (i.e., at 45 days), whereas for no-pretreatment runs, the content remained constant from the start to the middle and declined (from 36 to 34%) at the end of the experiment (i.e., at 90 days). We also report the chemical structure of microalgae with and without pretreatments.