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The present paper is a report of multiple mammary gland colonization in dairy cows caused by bacteria before and 6 days after the end of an 8-day effective mastitis therapy with the use of a purely herbal medicine, which is in line with the European Green Deal (EGD) and the farm to fork (F2F) strategy program to reduce antibiotic use in farming. The microorganisms were isolated from milk samples from 45 cows of the native Polish Black and White (ZB) breed kept in a farm in Southern Poland. For Staphylococcus aureus isolates additional biochemical tests and genetic analyses were performed. All isolated bacteria, often the same for all cows, were Gram-positive cocci, rods and coryneforms, while tests showed also the presence of yeasts; the results indicate that the microorganisms adapted to the bovine mammary gland can even spread from one animal to another in a farm. Counts of some bacteria and yeast species was observed to decrease after the end of a successful herbal ointment therapy of mastitis; nevertheless, some of the same species of bacteria and yeasts were present both before and after therapy. There were no obligate pathogens evidenced, but many commensal and environmental species of bacteria and yeasts were reported. There is important discrimination of commensal microorganisms from obligate pathogens in such investigations. This paper shows a positive effect of herbal ointment application, broadens our knowledge on primary colonization and may prove important for epidemiological investigations in cases of secondary mammary gland infections in cows. [ABSTRACT FROM AUTHOR]