Porphyromonas gingivalis is an important pathogen in the development of chronic periodontitis, a subgingival plaque biofilm-associated disease characterised by progressive loss of alveolar bone and soft tissue attachment to the tooth. Increase in subgingival plaque numbers of P. gingivalis in conjunction with Treponema denticola is a predictor of disease progression. In the mouse periodontitis model P. gingivalis at low numbers (<0.01% of the biofilm) can induce plaque dysbiosis, with increased plaque biomass and shifting the species composition to a pathogenicity-associated profile. This has been attributed to the hydrolytic activities of P. gingivalis proteinases RgpA, RgpB and Kgp, collectively called gingipains, and associated dysregulation of immune responses. The gingipain zymogens have unusually large, 25 kDa, N-terminal propeptides (PP) that are removed during extracellular activation. The fate in vivo of the cleaved PP fragments has not been determined. We show that the Arg-gingipain propeptides produced in Escherichia coli (rRgpA-PP and rRgpB-PP) inhibit RgpA and RgpB with nM Ki thus are promising therapeutics. Furthermore, application of rRgpA-PP and rRgpB-PP prevents the establishment of mono- and multispecies biofilm and importantly disrupts established mono- and multi-species biofilms in vitro. The anti-biofilm effect was found unexpectedly to be due to antimicrobial functions of rRgpA-PP and rRgpB-PP, including against P. gingivalis, via a mechanism independent from gingipain activities. However, rRgpA-PP and rRgpB-PP do not harm all species, including T. denticola, demonstrating selectivity of action. Gingipain propeptides may represent the first identified members of a novel class of bacteriocin, ones that derive from a larger precursor protein where in situ they have an alternative function. We propose that in vivo, the released gingipain propeptides via selective killing or growth inhibition could promote the observed pathogenic dysbiosis of the subgingival plaque biofilm observed in periodontal disease.