To discover new probiotics that can protect mammary glands from mastitis, 40 Lactobacillus (Ligilactobacillus) spp. isolates from bovine milk were subjected to a preliminary series of in vitro subtractive analyses. Antibiotic susceptibility testing was performed according to the ISO norm 10932. Many lactobacilli had elevated MIC values for kanamycin (35%), but fewer were resistant to chloramphenicol (15%), streptomycin (7.5%) and tetracycline (5%). The enzymic activities of lactobacilli were tested using an API ZYM system. Nearly 27% exhibited undesirable activities (β-glucuronidase, β-glucosidase and N-acetyl-β-glucosaminidase). The safe strains were monitored for antimicrobial activity against Staphylococcus aureus, Escherichia coli, Salmonella enteritidis, and Bacillus cereus using microtiter plates and for their ability to form biofilms using the crystal violet assay. The antimicrobial activity of lactobacilli against indicator bacteria ranged from 29 to 89% and the isolates exhibited moderate-to-high biofilm formation. Suitable strains were selected for whole-genome sequencing analysis. Antibiotic-resistance genes and putative virulence genes were not predicted in the genomic analysis. Moreover, the isolate Ligilactobacillus salivarius 48 carries genetic information responsible for bacteriocin production that is similar to that encoding salivaricin CRL1328. Our study demonstrates the safety of the above mentioned isolate, which has potential to be used as a probiotic, exerting health benefits through production of antimicrobial substances.