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Abstract Preventive infusion of antibiotics in the mammary gland of cows consumes 11 tons/year of medically relevant antimicrobials, yet, this practice might not be critical to prevent new infections in the healthy mammary gland of cows. Here, we used next-generation sequencing and quantitative real-time PCR to determine the impact of dry cow therapy without antibiotics on milk microbiome and bacterial load, respectively. Cows diagnosed as negative for mastitis at dry off were randomly allocated to receive antibiotic (intramammary ceftiofur hydrochloride) and teat sealant or just teat sealant. Firmicutes was the most abundant phylum, and Corynebacterium, Acinetobacter, and Staphylococcus, often involved in mastitis cases, were the most abundant genera across treatments and time. However, there were no effects of antimicrobial on milk microbiome and bacterial load. Bacterial load was greater at seven days postpartum than at dry off. Dry cow therapy based on teat sealant without antibiotics can be used with no detrimental impacts on milk microbiome and bacterial load in cows with a healthy mammary gland.