Among the outstanding questions about the emergence of human-controlled fire is the systematic recurrence between the geochemical remains of fire and its preservation in the archaeological record, as the use of fire is considered a technological landmark, especially for its importance in food cooking, defensive strategies, and heating. Here we report fossil lipid biomarkers associated with incomplete combustion of organic matter at the Valdocarros II site, one of the largest European Acheulean sites in Spain dated to marine isotopic stage (MIS) 8/7 (~ 245 kya) allowing a multiproxy analysis of human-controlled fire use. Our results reveal isolated cases of highly concentrated and diverse polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs (APAHs), along with diagnostic conifer-derived triterpenoids in two hearth-like archaeological structures. The presence of combustion byproducts suggests the presence of anthropogenic (controlled) fires at Valdocarros—one of the oldest evidence of fire use in Europe-in association with Acheulean tools and bones. Hominins possibly used fire for two main activities, as a means of defense against predators and cooking. Our results help to better delineate major gaps in our current knowledge of human-controlled fire in the context of the Middle-Pleistocene in Europe and suggest that human ancestors were able to control fire before at least 250 kya.