Many pathogenic bacteria can protect themselves from the effects of antibiotics and the host immune response system by forming biofilms. Eradication of bacterial biofilms typically requires much higher concentrations of antibiotics, than are normally needed to kill cultured planktonic cells, raising serious clinical concerns. In an attempt to try to prevent the formation of biofilms or to break up existing biofilms of pathogenic bacteria, herein we have used the standard crystal violet assay as well as the Calgary biofilm device, to test several lactoferrin- and lactoferricin-derived antimicrobial peptides for their antibiofilm activity against Pseudomonas aeruginosa PAO1. Our results reveal that the short bovine lactoferricin-derived RRWQWR-NH2 (20-25) hexapeptide has no activity against P. aeruginosa PAO1. Moreover, the longer human lactoferricin-derived peptide GRRRRSVQWCA (1-11) and the bovine lactoferrampin (268-284) peptide were also almost devoid of activity. However, several different 'mix-and-match' dimeric versions of the two lactoferricin-derived peptides proved quite effective at low concentrations in preventing the formation of biofilms, and in some cases, could even eradicate existing biofilm. Some of the longer lactoferricin-derived dimeric peptides acted through a bacterial killing mechanism, while others seemed to interfere in cellular signalling processes. Taken together, our results reveal that synthetic dimeric peptides made up of the short naturally-occurring human and bovine lactoferricin constructs, can be further developed as antibiofilm agents.