Microalgae have attracted significant attention in biofuel research over recent years because of their rapid photosynthetic growth rates and the high lipid content achieved with certain species and culturing conditions. Most past research has focused on high-lipid algal species and increasing the lipid content in order to extract these lipids for conventional transesterification to biodiesel. High-lipid algae and the associated culturing conditions have often resulted in lower biomass productivities, thus limiting the potential yield of algae-based biofuel. An alternative approach is to culture fast-growing low-lipid algae and then convert the resulting biomass into bio-crude oil through a hydrothermal liquefaction (HTL) process. This article presents results for producing bio-crude oil via HTL conversion of Chlorella pyrenoidosa, a common fast-growing green microalgae species with low lipid content. The products of the HTL conversion generally self-separate into three phases: a gaseous product, an aqueous phase product, and bio-crude oil product. For the purpose of this study, a toluene-soluble fraction of the raw oil product was defined as refined oil. The natural phase-separation of HTL products is highly advantageous for algal biofuel production because it minimizes energy input for dewatering. To help optimize the HTL process, the effects of key operating parameters (initial pressure, reaction temperature, and reaction time) on the oil product yields were investigated. The experimental results indicate that reaction temperature and reaction time can greatly affect the refined oil yield. The initial pressure provided by nitrogen had little effect on oil yield. The highest refined oil yield was 39.4% ±1.2% of the total dry mass of algal feedstock, which was achieved at 280°C reaction temperature with 120 min reaction time.