Chronic wound infection resulting from polymicrobial colonisation with biofilm-forming bacteria poses a major global threat to public health and patient welfare. The opportunistic pathogen Enterococcus faecalis is one of the most commonly isolated microbes from wound infection, frequently isolated in tandem with pathogenic species including Staphylococcus aureus. E. faecalis biofilm formation drastically increases its virulence potential, providing protection against antimicrobial treatments and immune system clearance. Neutrophils represent a key component of the innate immune system, making up ~70% of circulating leukocytes and are the first cells to be mobilised in response to infection. Currently, interactions between neutrophils and biofilm are poorly understood, particularly in the context of multi-species biofilm. In this study, we first identified that E. faecalis (planktonic) infection of primary murine neutrophils suppressed neutrophil cell death out to 24 post-infection (p.i.). This was in striking contrast to infection with S. aureus alone or a mixed (1:1) infection with E. faecalis and S. aureus which drove rapid cell death. Whilst a 24 h biofilm of E. faecalis or S. aureus also suppressed or accelerated neutrophil cell death, respectively, a polymicrobial biofilm of the two species resulted in an intermediate phenotype. Confocal imaging with fluorescent bacterial strains revealed that murine neutrophils were capable of engulfing and clearing E. faecalis biofilm-associated bacteria, and indeed succumbed rapidly to a biofilm of S. aureus. In the polymicrobial biofilm, we observed the formation of homogeneously mixed microcolonies of E. faecalis and S. aureus. We next quantified the ability of neutrophils to clear these 24 h single- and multi-species biofilms via biomass (via crystal violet) and colony forming units (CFU). Current work seeks to examine the cytokine responses of neutrophils to planktonic versus biofilm-associated E. faecalis and S. aureus single and mixed species interactions. Our current evidence points to the hypothesis that the formation of a polymicrobial biofilm enables collaborative persistence of E. faecalis and S. aureus through immunomodulation of neutrophils, facilitating chronic infectious disease.