Bovine mastitis is an inflammation of the mammary gland generally due to an intramammary infection. Prophylactic treatments mostly rely on post-milking teat disinfection with chemical products such as iodine. However, the latter are questioned due to a possible irritation of the teat skin but also dissemination of residues in milk and environment. Besides, the antibiotics commonly used to treat mastitis are unfortunately not entirely effective and may contribute to the risk of dissemination of antimicrobial resistance, inviting us to explore alternative solutions. Here, a microbial strategy to prevent mastitis was explored, aiming to improve the barrier effect of the teat microbiota, through the application of a lactic acid bacteria (LAB) strain. Firstly, the impact of Lacticaseibacillus paracasei CIRM BIA 1542 application on the teat skin was explored on 23 Holstein cows in mid-lactation. Treatment (LAB, iodine or no treatment) was applied twice a day post-milking on the 4 quarters of healthy animals for 15 days. Blood, milk samples and teat skin swabs were collected at D1, D8, D15 and D26 to evaluate the LAB treatment impact at the microbial, immune and physiological levels. Data were analyzed using an analysis of variance based on mixed models. L. paracasei CIRM BIA 1542 was transiently present on teat skin and in foremilk during the 15 days of treatment, but not in the cisternal milk. Total microbial population on teat skin, in foremilk and cisternal milk was significantly increased in LAB-treated cows compared with iodine-treated cows (p < 0.05). However, no pathogen was found in cisternal milk. Ongoing analyses will evaluate the impact on teat skin and in foremilk microbiota using metataxonomic. In addition, LAB treatment did not trigger any major inflammatory response in the mammary gland: no significant impact was observed on milk somatic cell scores, although interleukin 8 released in milk tended to be slightly higher in LAB-treated cows compared with others. Finally, LAB treatment had no impact on the functionality and the integrity of the mammary epithelium, as revealed by no significant effect on milk yield and composition, nor on the mammary epithelial cell exfoliation rate into milk or the milk Na+:K+ ratio. Altogether, these results indicate that a topical treatment with L. paracasei CIRM BIA 1542 is safe with regard to mammary gland physiology and immune system, while impacting its microbiota, inviting us to further explore its effectiveness for mastitis prevention.