Milk fat content and its fatty acid (FA) profile are important components of both the milk nutritional and technological qualities. The present study was conducted to evaluate the influence of calf presence during milking and of cow breed on milk yield, gross composition, FA profile and milk lipolytic system. A total of 30 Prim’Holstein (PH) and Salers (S) primiparous lactating cows were milked in the presence or absence of their calves during 210 days of lactation and bulk milks were collected during 6 weeks in a restricted part of the whole experimental period for analyses. Calf presence increased milk yield and decreased the milk fat content in S cows, whereas no difference was observed for the PH cows. The PH milk had higher levels of short- and medium-chain FA and C18:2 n−6, whereas S milk had higher levels of C18:2 cis-9,trans-11 (conjugated linoleic acid, CLA), C18:3 n−3 and C20:5 n−3. The S milk also had higher initial lipolysis. Calf presence during milking increased the C16:0 percentage and decreased C18:0, C18:2 n−6, C18:3 n−3, total polyunsaturated FA percentages in milk fat. Calf presence decreased the milk trans-C18:1 level only in S cows. Cis-9,trans-11-CLA level increased in the calf presence in PH cows, whereas it decreased in S cows. Calf presence during milking increased milk lipolysis after 24 h only in the PH cows. Calf presence during milking had significant effects on the milk yield, gross and fine composition, and lipolysis but these effects differed according to the breed.