The increasing incidence of infections with multi-drug resistant Enterococcus faecium necessitates studies to increase knowledge on the pathogenesis of these infections. In this study, the contribution of peritoneal macrophages during E. faecium peritonitis was investigated. In an ex vivo setting, peritoneal macrophages harvested from C57BL/6 mice were responsive to, and able to phagocytose and kill, E. faecium. In vivo, peritoneal macrophages were depleted by intraperitoneal injection of clodronate-encapsulated liposomes, prior to inducing E. faecium peritonitis. Depletion of resident peritoneal macrophages caused a clear delay in peritoneal clearance of E. faecium with increased systemic dissemination. Mice depleted of peritoneal macrophages were able to recruit macrophages and neutrophils to the peritoneal cavity after infection, comparable to control mice. Furthermore, increased levels of peritoneal cytokines and chemokines were found in mice depleted of peritoneal macrophages. This study indicates that peritoneal macrophages are important in the early containment of E. faecium peritonitis and for the regulation of the inflammatory response.