Simple SummaryAlbeit being crucial for survival of offspring, milk in the canine species is relatively poorly investigated. Normal milk microbiota is a proven and important source of bacteria able to colonize the gut of newborns described in several species, including humans. Scientists now consistently find a range of bacterial species in milk from healthy individuals lacking any clinical signs of mastitis. To elucidate this matter, we analyzed milk of healthy dams who delivered healthy puppies naturally and investigated whether it contains bacteria using standard culture media for aerobic and anaerobic growth of microorganisms. Results from 210 samples collected twice with an interval of 7 days in the early postpartum period reportedStaphylococcaceaeandEnterococcaceaeto be most represented. No significant difference in bacterial growth from milk sampled first or second time was noted. Every dam had bacteria in most mammary glands, and only 14% of total samples had no bacterial growth. The composition of bacteria from all milk samples did not differ significantly, indicating consistent occurrence of bacteria in normal, healthy milk, not mere contamination. Considering the global threat posed by antibiotic resistance, their application should be avoided in individuals without additional clinical signs of inflammation.AbstractStudies of microbiota in normal canine milk from healthy dams are sparse. As is the case with blood and urine, it was considered that milk contains no microbiota. Any discovery of bacteria in canine milk is, therefore, often noted to be a result of contamination during sampling or interpreted as mastitis and treated with antibiotics. Milk was collected twice within 19 days after natural parturition from 11 lactating dams, with no general or local clinical signs of mastitis or other disease. The skin and teats were prepared with an antimicrobial protocol prior to each milk sampling. In total, 210 milk samples were collected and assessed for a number of bacterial colonies grown on each plate. Bacterial growth was detected in 180 samples (86%).Staphylococcus pseudintermedius,Enterococcusspp.,Clostridiumspp., Coagulase-Negative Staphylococci (CoNS),Streptococcusspp.,Streptococcus canis,Bacillusspp.,Pasteurellaspp., andEscherichia coliwere identified from pure and/or mixed bacterial growth, listed in descending order of occurrence. Despite the small sample size, the consistent occurrence of bacteria in early postpartum dams indicates a genuine occurrence of bacteria in canine milk, rather than random contamination. The finding of bacteria in the milk of dams should not, therefore, be the sole argument for the diagnosis of mastitis.