Women use a variety of practices to handle and store their pumped breast milk. Little is known about how these practices influence milk’s bacterial composition. We conducted an in-home randomized, controlled, crossover trial and an observational substudy within it. Fifty-two women pumped the same breast twice within 3.5 h, once with their own pumps and collection kits (“own” supplies) and once with a multiuser pump and sterile collection kits (“sterile” supplies). The type of pump supplies used first was randomized. Milk samples were separated into sterile containers and processed within 2 h after expression or after storage in home refrigerators for 2 or 4 d, or in home freezers for up to 30 d with or without refrigeration for the first 4 d. Before women pumped with their own supplies, the following areas were swabbed: areolas, women’s hands, collection kits, pump tubing, and infants’ mouths. Bacterial composition was assessed via aerobic culturing (milk only) and high-throughput sequencing of the V1-V3 region of the 16S rRNA gene from DNA extracted from samples (milk and swabs). Women’s pumping practices were recorded. Culture and 16S analyses revealed the bacterial composition of milk collected with women’s own supplies contained several times more live aerobic and gram-negative bacteria, and >7 times more Proteobacteria than milk collected with sterile supplies. The majority of these extra bacteria likely originated from milk collection kits and survived refrigeration, freezing, or both better than native milk bacteria (bacteria in milk collected with sterile supplies). More rigorous cleaning practices (e.g., sterilization versus washing or rinsing) were associated with significantly fewer bacteria in milk collected with women’s own supplies, including several times lower odds of milk being culture-positive for gram-negative bacteria. This research revealed typical real-life pumping practices changed the bacterial composition of breast milk, most notably by adding live gram-negative bacteria and Proteobacteria. The majority of these extra bacteria likely originated from milk collection kits and survived storage better than native milk bacteria. These findings can contribute to the development of evidence-based recommendations for handling and storing pumped milk pumped. More research is needed to understand the implications of these findings for infant and maternal health.