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To evaluate the in vitro bactericidal activity of the novel antimicrobial peptide (AP) CECT7121 against Gram-positive bacteria from mastitic dairy cattle.A total of 15 Staphylococcus aureus, 10 Streptococcus dysgalactiae, 7 Strep. uberis, 1 Strep. agalactiae strains were isolated from 33 different mastitic dairy cattle, sourced from two dairies in Tandil-Argentina. Isolates from each of the bacterial species screened which developed the lowest inhibition zones in response to the peptide, were further evaluated in a series of time-killing curve studies. No survivors were detected in whole strains (from the three Streptococcal species isolated) within 120 min of incubation in presence of the peptide. The Staph. aureus isolates were less sensitive but, nevertheless, a drop in viable counts to below the detection limit was achieved for all the test strains by the final postincubation sampling point at 180 min.The study demonstrated the in vitro efficacy of the AP-CECT7121 against a variety strains of Gram positives isolated from mastitic dairy cattle.There is urgent global interest in the development of natural alternatives for the control and prevention of mastitis. Confirmation of the in vitro activity of the novel AP-CECT7121 against Gram-positive isolates encourages further research.