Infections by opportunistic pathogens can lead to lactational mastitis, characterized by duct obstruction and painful inflammation in the mammary gland. Sub-acute mastitis (SAM) is a less severe state of the disease, but it is extremely prevalent among lactating women, being one of the main reasons for early weaning. Although the etiology and diagnosis of acute mastitis is well established, very little is known about the underlying mechanisms causing the sub-acute state. Milk samples were obtained from healthy and SAM-suffering mothers at two time points (during the mastitis and when the symptoms disappeared). Total (DNA-based) and active (RNA-based) bacterial composition were analysed by 16S rRNA Illumina sequencing and bacterial load was estimated by qPCR. Bacterial load was significantly higher in the mastitis samples compared to healthy mothers and decreased after the clinical symptoms disappeared. Bacterial diversity significantly decreased in milk from SAM samples. Significant differences in bacterial composition and activity were found in milk samples from healthy and SAM mothers, and possible causing agents included not only Staphylococci but also several Streptococci and oral bacteria. Contrary to acute mastitis, the same bacterial species were found in samples from healthy and diseased mothers, although at different proportions. Finally, mammary epithelial cell lines were exposed to human milk from healthy and SAM mothers, showing an over-production of the inflammatory marker IL8 in the presence of SAM bacteria. Our work therefore supports that SAM has a bacterial origin, with increased bacterial loads, reduced diversity and altered composition, which were partly recovered after the treatment. Taken together, the data suggest a polymicrobial and varying etiology of SAM, which appears to be the outcome of a bacterial dysbiosis.