Background and Aim: Mastitis is one of the most important diseases of cows and the most expensive pathology for the dairy industry. Therefore, this study was conducted to explore the role of microorganisms isolated from cows with mastitis in the formation of biofilms under the conditions of farm biogeocenosis in the Moscow region. Materials and Methods: Periodic visits to 12 farms in the Moscow region were conducted to explore the microbial profile of the udder of cows with mastitis. During the visits, 103 milk samples from sick animals were collected and examined. Through microbiological analyses, 486 cultures of microorganisms were identified, which are assigned to 11 genera. Mastitis in cows is caused not only by a single pathogen but also by microbial associations, which included two to seven microbial isolates. Results: It was observed that 309 isolates (63.6%) from the total number of isolated microorganisms could form a biofilm. The ability to form biofilms was most frequently observed in Staphylococcus aureus (18.8%), Escherichia coli (11.9%), and Staphylococcus uberis (11.7%) cultures from the total number of biofilm-forming microbial cultures. Low biofilm-forming ability among the isolated microorganisms was found in lactobacilli, wherein only 20 (22.5%) Lactobacillus strains had the ability to form biofilms. The isolated microorganisms exhibited different sensitivities to antimicrobial agents, which cause difficulty in selecting an antimicrobial agent that would act on all aspects of the parasitocenosis. Conclusion: A high proportion of microorganisms isolated from cows with mastitis have the ability to form biofilms. The isolated microorganisms exhibited different and highly heterogeneous sensitivity to the action of antimicrobial drugs. This causes difficulty in using these tools for the effective control of mastitis in cows, which is frequently caused by pathogenic associations of microbial biofilms. Therefore, it is important to explore novel and more effective methods to combat this disease. Copyright: Rudenko, et al. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.