This work investigated an influence of operating conditions on the biocrude yield and properties obtained from hydrothermal liquefaction (HTL) of Coelastrum sp. microalgae in a two-step sequential HTL (THTL) and a single-step HTL (SHTL) using a semi-continuous system. A higher biocrude yield with a lower nitrogen content was obtained with the THTL process than the SHTL one. The operating temperature, pressure and water flow rate were sequentially varied in a univariate analysis for a 2 h reaction time to optimize the obtained biocrude yield. Increasing the temperature improved the biocrude yield, but the second step temperature should not be higher than 320 °C to prevent the thermal cracking to gaseous compounds. The optimal conditions of THTL were preliminarily temperature of 200 and 320 °C and pressure of 7 and 20 MPa for the first and second step, respectively, both with a water flow rate of 0.50 mL/min.