Bovine mastitis is a major disease in the dairy industry. The current approaches are not entirely effective and may contribute to the risk of dissemination of antimicrobial resistance, supporting the need for alternative treatments. Here, a microbial strategy aiming to shape the teat microbiota towards a protective one through the application of a lactic acid bacteria (LAB), was explored on 23 Holstein cows in mid-lactation. Treatment (Lacticaseibacillus paracasei CIRM BIA 1542 sprayed at ~1.109 CFU/mL (L; n = 8), iodine (I; n = 7) or no treatment (N; n = 8)) was applied post-milking twice a day on the 4 teats of healthy animals for 15 days. Blood and milk samples and teat skin swabs were collected at D1, D8, D15 and D26 before morning milking and at D15 before evening milking (D15e) to evaluate the LAB treatment impact at the microbial (presence of pathogens, microbiota analysis), immune (somatic cell count) and physiological levels (milk production, markers of mammary epithelium integrity). Data were analysed using an ANOVA with day and treatment as fixed effects and cow as a random effect. L. paracasei was transiently present on teat skin and in foremilk during treatment, as illustrated by a LAB population in foremilk 1.5 and 29-fold higher in L than I at D15 and D15e, respectively (p < 0.01). The total microbial population in cisternal milk, foremilk and on teat skin was significantly higher during treatment (p < 0.05) in L (1.79-4.84 log (CFU/mL)) compared with I (1.01-3.95 log (CFU/mL)). However, no pathogen was found in cisternal milk. Besides, LAB treatment did not trigger any major inflammatory response in the mammary gland (no significant impact on milk somatic cell scores) and had no impact on the functionality and the epithelium integrity (no significant impact on milk yield and composition, mammary epithelial cell exfoliation rate into milk or Na+:K+ ratio). Altogether, these results indicate that LAB treatment is safe with regard to mammary gland functionality and immune system while impacting its microbiota.