Attempts to deal with the problem of detrimental biofilms using nanoparticle technologies have generally focussed on exploiting biocidal approaches. However, it is now recognised that biofilm matrix-components may be targets for the disruption or dispersion of biofilms. Here, we show that the functionalization of gold nanoparticles with the enzyme, proteinase-K (PK) led to both biocidal and matrix disruption effects within Pseudomonas fluorescens biofilms and released cells. This study highlights the potential mechanisms underpinning the properties of Proteinase-K functionalized gold nanoparticles. With the emergence of biocide-resistant biofilm-forming organisms, novel nanoparticle strategies may provide the ideal solution for disrupting and inactivating biofilm cells, thereby minimising the use of biocides or antibiotics.