Archaeal sequences have been detected in human colostrum and milk, but no studies have determined whether living archaea are present in either of these fluids. Methanogenic archaea are neglected since they are not detected by usual molecular and culture methods. By using improved DNA detection protocols and microbial culture techniques associated with antioxidants previously developed in our center, we investigated the presence of methanogenic archaea using culture and specific Methanobrevibacter smithii and Methanobrevibacter oralis real-time PCR in human colostrum and milk. M. smithii was isolated from 3 colostrum and 5 milk (day 10) samples. M. oralis was isolated from 1 milk sample. For 2 strains, the genome was sequenced, and the rhizome was similar to that of strains previously isolated from the human mouth and gut. M. smithii was detected in the colostrum or milk of 5/13 (38%) and 37/127 (29%) mothers by culture and qPCR, respectively. The different distribution of maternal body mass index according to the detection of M. smithii suggested an association with maternal metabolic phenotype. M. oralis was not detected by molecular methods. Our results suggest that breastfeeding may contribute to the vertical transmission of these microorganisms and may be essential to seed the infant's microbiota with these neglected critical commensals from the first hour of life.