A hydrothermal pretreatment of the microalga Nannochloropsis gaditana at mild temperatures was studied in order to reduce the N and O content in the biocrude obtained by hydrothermal liquefaction (HTL). The work focused on the evaluation of temperature, reactor loading, and time (factors) to maximize the yield of the pretreated biomass and the heteroatom contents transferred from the microalga biomass to the aqueous phase (responses). The study followed the factorial design and response surface methodology. An equation for every response was obtained, which led to the accurate calculation of the operating conditions required to obtain a given value of these responses. Temperature and time are critical factors with a negative effect on the pretreated biomass yield but a positive one on the N and O recovery in the aqueous phase. The slurry concentration has to be low to increase heteroatom recovery and has to be high to maximize the pretreated microalga yields. Response equations were obtained for the analyzed responses, which facilitated the accurate prediction of the operating conditions required to obtain a given value of these responses.