Investigation of pathogen-host interactions on a molecular level requires sophisticated in vitro infection procedures, especially in the presence of different pathogens.Super-infections of influenza viruses (IV) and bacteria, with increasing incidence of Staphylococcus aureus (S. aureus) cases, are a long-known phenomenon and represent a major complication in IV-infected patients. Although several in vivo studies have improved our knowledge about pathogenesis and immune responses of super-infections that result in increased morbidity and mortality, the consequences of the direct interplay of viruses and bacteria on a molecular level in affected cells that may contribute to the deadly synergism of these pathogens are so far poorly characterized. Here we describe different infection schemes to study IV and S. aureus coinfections of distinct cell populations in vitro. Depending on the focus of interest, regulation of cell responses such as signalling mechanisms or pro- and anti-inflammatory cytokine expression, or consequences for the viral or bacterial life cycle, can be analyzed. The described infection procedures could be used as guidelines and adapted to super-infection settings of other viral and bacterial pathogens.