Bacterial pleural infections, especially those caused by Staphylococcus aureus, are associated with high mortality and are complicated by biofilm formation. Due to the human-specific nature of S. aureus, rodent models are insufficient for research. This study investigated the effects of S. aureus infection on human pleural mesothelial cells (HPMC) using a newly established 3D organotypic co-culture model derived from human tissue, incorporating human pleural fibroblasts (HPFs) as the basal layer. Infected samples were collected at defined time points for histological analysis and immunostaining of tight junction proteins, revealing changes comparable to in vivo empyema. Cytokine measurements confirmed host-pathogen interactions, and mesothelial cells produced VEGF at physiological levels. In contrast, sterile controls showed intact, viable cells. This novel model offers a promising in vitro tool to study biofilm formation and host responses in pleural empyema.