In contrast to classical, oleaginous biomasses, microalgae are usually characterized by an increased water content and a relatively simple, albeit very robust, structure. Both properties represent a significant challenge for established processes. Consequently, an alternative method for lipid extraction from microalgae was investigated in this work: Extraction with liquefied dimethyl ether (DME). Therefore, the solvent properties of DME, known uses, and existing (regulatory) framework were examined in depth. Batch extractions of four different freeze-dried microalgae (Arthrospira platensis, Nannochloropsis gaditana, Phaeodactylum tricornutum, and Scenedesmus almeriensis) were performed to assess the influence of algae pre-treatment and species on crude oil yields and fatty acid extraction with DME. Follow-up experiments with P. tricornutum revealed that the water content of the biomass plays a crucial role in the extraction. The presented work demonstrated that lipid extraction with liquefied DME is a promising unit operation for algae-biorefineries. In the case of P. tricornutum, energy consuming drying or alternative feedstock pretreatment is not required. This fundamental advantage could play a pivotal role for the competitiveness of microalgae-based production in the future.