Owing to its ability to form biofilms, Staphylococcus aureus is responsible for an increasing number of infections on implantable medical devices. The aim of this study was to develop a mouse model using microbeads coated with S. aureus biofilm to simulate such infections and to analyse the dynamics of anti-biofilm inflammatory responses by intravital imaging. Scanning electron microscopy and flow cytometry were used in vitro to study the ability of an mCherry fluorescent strain of S. aureus to coat silica microbeads. Biofilm-coated microbeads were then inoculated intradermally into the ear tissue of LysM-EGFP transgenic mice (EGFP fluorescent immune cells). General and specific real-time inflammatory responses were studied in ear tissue by confocal microscopy at early (4-6h) and late time points (after 24h) after injection. The displacement properties of immune cells were analysed. The responses were compared with those obtained in control mice injected with only microbeads. In vitro, our protocol was capable of generating reproducible inocula of biofilm-coated microbeads verified by labelling matrix components, observing biofilm ultrastructure and confirmed in vivo and in situ with a matrix specific fluorescent probe. In vivo, a major inflammatory response was observed in the mouse ear pinna at both time points. Real-time observations of cell recruitment at injection sites showed that immune cells had difficulty in accessing biofilm bacteria and highlighted areas of direct interaction. The average speed of cells was lower in infected mice compared to control mice and in tissue areas where direct contact between immune cells and bacteria was observed, the average cell velocity and linearity were decreased in comparison to cells in areas where no bacteria were visible. This model provides an innovative way to analyse specific immune responses against biofilm infections on medical devices. It paves the way for live evaluation of the effectiveness of immunomodulatory therapies combined with antibiotics.