Human milk contains a low-biomass, low-diversity microbiome, consisting largely of bacteria. This community is of great research interest in the context of infant health and maternal and mammary health. However, this sample type presents many unique methodological challenges. In particular, there are numerous technical considerations relating to sample collection and storage, DNA extraction and sequencing, viability, and contamination. Failure to properly address these challenges may lead to distortion of bacterial DNA profiles generated from human milk samples, ultimately leading to spurious conclusions. Here, these technical challenges are discussed, and various methodological approaches used to address them are analyzed. Data were collected from studies in which a breadth of methodological approaches were used, and recommendations for robust and reproducible analysis of the human milk microbiome are proposed. Such methods will ensure high-quality data are produced in this field, ultimately supporting better research outcomes for mothers and infants.