Background: In vitro investigations of human innate immune responses to extracellular bacteria commonly utilise killed preparations in preference to live organisms. The effects of the bacterial preparation method on the activation of innate signalling pathways by the common opportunistic pathogen Staphylococcus epidermidis (SE) are unknown. Materials and methods: Mononuclear cell cytokine expression patterns induced by live (LSE), heat-killed (HKSE) and ethanol-killed SE (EKSE) were characterized at the transcriptional and translational level. Toll-like receptor (TLR)-activating capacity of the preparations was analysed using TLR-transfected human embryonic kidney cells. Results: Live SE activated NF-κB, STAT1, type I interferon, and inflammasome pathways. Killed preparations engaged the NF-κB pathway, but had significantly lower capacity to activate other innate immune pathways. Conclusions: Killing of extracellular bacteria has significant qualitative and quantitative effects on key aspects of innate responses in vitro. Interpretation of in vitro data and extrapolation of findings should take into account the potential effects of bacterial preparation and should not assume that responses to killed bacteria are predictive of responses to live organisms.