Human milk is a low-biomass fluid whose bacterial community may contribute to infant gut colonization; however, the milk microbiota of women from southern Peru remains uncharacterized. This study aimed to describe the bacterial composition and diversity of human milk at one month postpartum (days 30–33 of lactation) in women from Arequipa, Peru, using full-length 16S rRNA gene nanopore sequencing. In this cross-sectional study, milk was collected from 29 mothers, and 21 samples were analyzed after excluding eight with a relative abundance of Enterobacterales ≥30%. The microbiota was dominated by the phylum Bacillota (formerly Firmicutes; mean 92%), with Streptococcus (62.9%) and Staphylococcus (21.8%) forming the microbial core. Presumptive species-level assignments corresponded mainly to oral and skin commensals (Streptococcus mitis, Streptococcus salivarius, and Staphylococcus epidermidis). Alpha diversity was low (medians: Shannon index 0.66; Simpson index 0.30; observed richness 11 genera). In the principal coordinates analysis (Bray–Curtis), samples clustered according to their dominant genus. Observed richness was nominally higher in mothers with normal body mass index (p = 0.0071), whereas overall community composition was not associated with maternal, obstetric, or dietary variables (PERMANOVA, all p ≥ 0.073). This study provides the first characterization of the human milk microbiota in women from Arequipa, establishing a baseline for future studies.