The microbiome of bovine milk has been characterized in several studies. About 53% of dairy operations identify coliforms in mastitis cases, and ceftiofur accounts for over 50% of treatments of mastitis in the USA. Nevertheless, the efficacy of Escherichia coli mastitis treatment is still ambiguously discussed. Our objectives were (1) describe the milk microbiome before, during, and after experimental infection, clinical disease, and treatment; and (2) evaluate the effect of extended treatment with ceftiofur in infected quarters and ipsilateral healthy quarters. Holstein cows (n = 12) were infected with 100 cfu of a characterized E. coli strain in one quarter. Treatment was randomly assigned at the animal level. Three quarters were sampled in each cow: the challenged quarter, an ipsilateral unchallenged quarter included in the same treatment group, and a third quarter which did not undergo any intervention. Thirty-six samples were collected from −72 to 216 h relative to challenge. Treated animals received 5 IMM infusions of ceftiofur at 24-h intervals in both challenged and non-challenged quarters. The first IMM ceftiofur treatment was administered 48 h after infection. Infection was confirmed through cfu and RAPD-PCR. DNA was extracted from milk, the 16S rRNA gene was amplified, sequenced in an Illumina Mi-Seq, and analyzed with QIIME. Weighted Unifrac distances were analyzed in QIIME, ANOSIM was performed on R, and mixed models were built in SAS to evaluate the effect of treatment. Intramammary challenge with E. coli severely affected microbial diversity and increased levels of Enterobacteriaceae. No treatment effect was observed in the microbial profile, pathogen load, or diversity of challenged quarters. Challenged quarters returned to a healthy milk microbial profile by the end of the study period independent of treatment. No effect of intramammary infusion of ceftiofur was observed in the microbiome of healthy quarters. In conclusion, our results indicate that the mammary gland harbors a resilient microbiome, capable of reestablishing itself after experimental infection with E. coli independent of antimicrobial treatment.