Bovine lactoferricin is an antimicrobial, cationic peptide generated upon gastric pepsin cleavage of bovine lactoferrin. We investigated the bactericidal effects of native lactoferricin [Lfcin B(17-41)], a shortened derivative [Lfcin B(17-31)] and the all-D-amino acid counterpart of Lfcin B(17-31) against Escherichia coli and Staphylococcus aureus. The results revealed different activities for the peptides against Gram-positive and -negative bacteria. D-Lfcin B(17-31) was the most efficient peptide against E. coli. The same peptide showed improved activity against S. aureus, D-Lfcin B(17-31) showed a significant better efficacy when compared to the L-form, but not when compared to Lfcin B(17-41). There was no correlation between the bactericidal concentrations and the time needed to achieve maximum effect. This indicates the importance of structural differences between the peptides and/or bacteria and implies that the simple thesis of I antibacterial target is not valid for lactoferricin.