Recent studies report the presence of fungal species in breast milk of healthy mothers, suggesting a potential role on infant mycobiome development. In the present work, we aimed to determine whether the healthy human breast milk mycobiota is influenced by geographical location and mode of delivery, as well as investigate its interaction with bacterial profiles in the
same samples. A total of 80 mature breast milk samples from 4 different countries were analysed by Illumina sequencing of the ITS1 region, joining the 18S and 5.8S regions of the
fungal rRNA region. Basidiomycota and Ascomycota were found to be the dominant phyla, with Malassezia and Davidiella being the most prevalent genera across countries. A core formed by Malassezia, Davidiella, Sistotrema and Penicillium was shared in the milk samples from the different origins, although specific shifts in mycobiome composition were associated with geographic location and delivery mode. The presence of fungi in the breast milk samples was further confirmed by culture and isolates characterization, and fungal loads were estimated by qPCR targeting the fungal ITS1 region. Co-occurrence network analysis of bacteria and fungi showed complex interactions that were influenced by geographical location, mode of delivery, maternal age and pre-gestational Body Mass Index. The presence of a breast milk mycobiome was confirmed in all the samples analysed, regardless of the geographic origin.