Dedicated lactation rooms are a modern development as mothers return to work while still providing breastmilk to their absent infants. This study describes the built environment microbiome of lactation rooms and daycares, and explores the influence of temperature and humidity on the microbiome of lactation rooms. Sterile swabs were used to collect samples from five different sites in lactation rooms at University of California, Davis and from five different sites in daycares located in Davis, California. DNA from the swabs was extracted and the V4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Temperature and relative humidity data were collected on a subset of the lactation rooms. Sampled lactation rooms could be either dedicated lactation rooms or could also serve other functions (e.g., combined lactation room and restroom lounge). The majority of sequence reads were identified as belonging to family Moraxellaceae, with 73% of all reads included in analysis identified as an unknown species of Acinetobacter. Alpha diversity was analyzed using the Shannon index, while beta diversity was analyzed using unweighted and weighted UniFrac distance. The Jaccard distance was used to measure amount of change at sampling locations between time points for analysis of the impact of temperature and humidity on the microbiome. There were significant differences in the beta diversity of the microbiome of lactation rooms by room type. There were also significant differences in the beta diversity of the microbiome by sample collection location. There were no significant differences in either alpha or beta diversity associated with room temperature or humidity. Additional studies are needed to understand if the differences in lactation room type may result in differences in the breastmilk microbiome of milk collected in those rooms, and to what extent any such differences may influence the infant microbiome.