Evaluation of the bovine transcriptome using RNA-Sequencing (RNA-Seq) has made substantial impact in assessing functional and structural genomes in cattle. A preliminary study evaluated the metatranscriptome of bovine milk to determine the composition and structure of bacterial populations influencing subclinical mastitis. Differences in bacterial presence in milk between healthy and mastitic quarters were found in Holstein cows using RNA-Seq technology. The objective of this study is to further evaluate the use of RNA-Seq technology to assess the non-mapped milk bacteria genome in dairy cattle. Transcriptomic and metagenomic analysis were performed using RNA-Seq technology and 16S ribosome sequencing on milk collected from 4 quarters of healthy (n = 4) and mastitic (n = 4) dairy cows. Milk samples were collected 3 h after morning milking to obtain a high percentage of epithelial cells. Cow teats were cleaned with gauze (70% isopropanol) and milk was collected by hand milking directly into sterile 50 mL Falcon tubes or using a 3 cm plastic cannula to collect milk within the teat canal to avoid external contamination. Total RNA was extracted from somatic cells (SC) and milk fat globule (MFG) membrane from both hand milking and cannula milk samples. Using a RNA-seq analysis pipeline, preliminary results revealed that 60 to 75% of reads were categorized as mapped to the bovine reference sequence. All reads not mapping to the bovine genome were annotated for MFG (32% hand milking, 20% cannula) and SC (25% hand milking, 12% cannula). Analysis of SC non-mapped reads identified differences in microbial species present in healthy and mastitic milk. Further analysis will lead to more precise mapping of sequence data and improved understanding of bacterial gene expression, integrating data generated from RNA-Seq and 16S sequencing. Future assessment of the non-mapped reads using RNA-Seq will be performed to study the ability of RNA-Seq technology to capture invasive pathogens in milk and their association to genes differentially expressed in healthy and mastitic quarters. This assessment may lead to a comparative approach to examine the immune response to infection in dairy cattle.