Bacterial pleural infections are associated with high mortality. Treatment is complicated due to biofilm formation. A common causative pathogen isStaphylococcus aureus(S. aureus). Since it is distinctly human-specific, rodent models do not provide adequate conditions for research. The purpose of this study was to examine the effects ofS. aureusinfection on human pleural mesothelial cells using a recently established 3D organotypic co-culture model of pleura derived from human specimens. After infection of our model withS. aureus, samples were harvested at defined time points. Histological analysis and immunostaining for tight junction proteins (c-Jun, VE-cadherin, and ZO-1) were performed, demonstrating changes comparable to in vivo empyema. The measurement of secreted cytokine levels (TNF-α, MCP-1, and IL-1β) proved host–pathogen interactions in our model. Similarly, mesothelial cells produced VEGF on in vivo levels. These findings were contrasted by vital, unimpaired cells in a sterile control model. We were able to establish a 3D organotypic in vitro co-culture model of human pleura infected withS. aureusresulting in the formation of biofilm, including host–pathogen interactions. This novel model could be a useful microenvironment tool for in vitro studies on biofilm in pleural empyema.