Microalgae are a promising biomass source because of their capability to fixate CO2 very efficiently. In this study, the potential of Microchloropsis salina biomass as a feedstock for fermentation was explored, focusing on biomass hydrolysis by employing various mechanical and chemical cell disruption strategies in combination with enzymatic hydrolysis. Among the mechanical cell disruption methods investigated on a lab scale, namely ultrasonication, bead milling, and high-pressure homogenization, the most effective was bead milling using stainless-steel beads with a diameter of 2 mm. In this way, 87–97% of the cells were disrupted in 40 min using a mixer mill. High-pressure homogenization was also effective, achieving 86% disruption efficiency after four passes on a 30–200 L scale using biomass with 15% (w/w) solids content. Enzymatic hydrolysis of the disrupted cells using a mixture of cellulases and mannanases yielded up to 25% saccharification efficiency after 72 h. Acidic hydrolysis of undisrupted cells followed by enzymatic treatment yielded around 30% saccharification efficiency but was coupled with significant dilution of the resulting hydrolysate. Microalgal biomass hydrolysate produced was determined to have ~8.1 g L−1 sugars and 2.5% (w/w) total carbon, as well as sufficient nitrogen and phosphorus content as a fermentation medium.