Aims: To isolate from the microbiota of the healthy cow udder commensal bacteria having antimicrobial activity against bovine mastitis pathogens, with a long-term view to their potential application as antimastitis probiotics. Methods and Results: Bacterial isolates from four healthy cow udders were tested for inhibitory activity against three Gram-positive indicator bacteria. This led to the selection of nine broadly inhibitory strains. All were of the Bacillus genus and their antimicrobial activities, which appeared heterogeneous on the basis of their antibacterial spectra and heat susceptibilities, enabled grouping of the inhibitory bacilli into six different inhibitory profiles. All displayed strong in vitro activity against Gram-positive mastitis pathogens. Inhibitory bacilli were recovered from each of the 11 udder samples collected over 7 months from one of these cows and the isolates included representatives of all six inhibitory profiles. Conclusions: Bacilli present in the udder microbiota of healthy cows can produce a variety of broadly active inhibitors of Gram-positive bacteria, including potential mastitis pathogens. Significance and Impact of the Study: Inhibitor-producing strains of commensal Bacillus species have been identified, which may have the potential for use as possible antimastitis probiotics.