Abstract Estuaries are considered as key habitats for the early life stages of fish. However, in the face of massive destruction of many estuarine intertidal areas, management and conservation measures are needed. Fish condition indicators may be used as a proxy of habitat quality and provide valuable information for management of coastal areas. In this study, the larvae of golden mullet ( Chelon auratus ) and European glass eels ( Anguilla anguilla ) were sampled in three sites of the Gironde Estuary. Different lipid classes and fatty acids were quantified: phospholipids (globally, phosphatidylethanolamine and phosphatidylcholine), triglycerides, omega‐3 (particularly docosahexaenoic and eicosapentaenoic acids), omega‐6 and C18:1. These biomarkers provide information on the nutritional status of the larvae as well as on prey availability and larvae diet between sites. One site significantly differed from the others as it seemed to offer abundant and better‐quality prey. The very high levels of omega‐3 contained in mullet larvae suggested that this site provided a high amount of diatoms. However, the mullet larvae that colonized this site also showed physiological stress that could be explained by exposure to pollutants through their prey. This work constitutes an essential baseline for developing biomarkers to assess the quality of habitats in a global change context.