Four microalgae species (Rhodomonas salina, Thalassiosira weissflogii, Thalassiosira pseudonana and Pavlova lutheri) were evaluated to estimate their potential as food for Ostrea edulis (L.) reproductive conditioning. Best ingestion and absorption were observed with R. salina (3.44 and 1.59 mg g− 1 h− 1, respectively), followed by T. pseudonana (2.75 and 0.98 mg g− 1 h− 1) and P. lutheri (2.40 and 0.91 mg g− 1 h− 1). Oysters fed T. weissflogii exhibited the lowest ingestion and absorption values (1.40 and 0.68 mg g− 1 h− 1). Proximate composition (proteins and carbohydrates) and lipid content (fatty acids and sterols) analysed in four main tissues (gonad, digestive gland, muscle and gills) also differed significantly with diet. Protein ranged from 355 mg g− 1 in the gonad of oysters fed P. lutheri to 837 mg g− 1 in gills of oysters fed T. weissflogii; whereas carbohydrates ranged from 17.5 mg g− 1 in gills of oysters fed P. lutheri to 271 mg g− 1 in gonads of oysters fed R. salina. An overall poor enrichment in total PUFAs across all diets masked some of their potential impact on nutrition. In gonad, however, the major polyunsaturated fatty acids (polar lipid fraction) were EPA (≈ 19% for oysters fed T. weissflogii and 14% for those fed P. lutheri) and DHA (17% for oysters fed P. lutheri and 15% for those fed R. salina). Sterol contents showed a clear transfer from food to oyster tissues except with P. lutheri, from which neither methylpavlovol nor ethylpavlovol (characteristic of Pavlophyceae) were detected in oyster tissues. Histological analysis showed that gametogenesis was active in oysters fed R. salina and T. weissflogii, whereas only low gonadic development occurred in unfed oysters or those fed P. lutheri. R. salina is accordingly highly recommended for O. edulis broodstock conditioning whereas P. lutheri should be excluded. Highlights ► We study the feeding assessment of four single microalgal species by flat oyster ► Best ingestion and absorption were observed with R. salina or T. weissflogii ► In gonad, the major polyunsaturated fatty acids (polar lipids) were EPA and DHA ► A transfer of sterols from food to oyster tissues occurred except with P. lutheri ► Gametogenesis was very active in oysters fed R. salina or T. weissflogii