Wound healing is a central physiological process that restores the barrier properties of the skin after injury, comprising close coordination between several cell types (including fibroblasts and macrophages) in the wound bed. The complex mechanisms involved are executed and regulated by an equally complex, reciprocal signalling network involving numerous signalling molecules such as catabolic and anabolic inflammatory mediators (e.g., cytokines, chemokines). In chronic wound environments, the balance in the molecular signatures of inflammatory mediators is usually impaired. Thus, we compared the ability of a collagen-based wound matrix against a synthetic wound matrix to attract fibroblasts and macrophages that deliver these signalling molecules. In particular, the balance between pro- and anti- inflammatory cytokine secretion was assessed. We found that the natural collagen-based matrix was the most efficient adhesive substrate to recruit and activate fibroblasts and macrophages on its surface. These cells secreted a variety of cytokines, and the natural biomaterial exhibited a more balanced secretion of pro- and anti-inflammatory mediators than the synthetic comparator. Thus, our study highlights the ability of native collagen matrices to modulate inflammatory mediator signatures in the wound bed, indicating that such devices may be beneficial for wound healing in the clinical setting.