During mucosal colonization, epithelial cells are concurrently exposed to numerous microbial species. Epithelial cytokine production is an early component of innate immunity and contributes to mucosal defence. We have previously demonstrated a synergistic response of respiratory epithelial cells to costimulation by two human pathogens, Streptococcus pneumoniae and Haemophilus influenzae. Here we define a molecular mechanism for the synergistic activation of epithelial signalling during polymicrobial colonization. H. influenzae peptidoglycan synergizes with the pore‐forming toxin pneumolysin from S. pneumoniae. Radiolabelled peptidoglycan enters epithelial cells more efficiently in the presence of pneumolysin, consistent with peptidoglycan gaining access to the cytoplasm via toxin pores. Other pore‐forming toxins (including anthrolysin O from Bacillus anthracis and Staphylococcus aureusα‐toxin) can substitute for pneumolysin in the generation of synergistic responses. Consistent with a requirement for pore formation, S. pneumoniae expressing pneumolysin but not an isogenic mutant expressing a non‐pore‐forming toxoid prime epithelial responses. Nod1, a host cytoplasmic peptidoglycan‐recognition molecule, is crucial to the epithelial response. Taken together, these findings demonstrate a role for cytosolic recognition of peptidoglycan in the setting of polymicrobial epithelial stimulation. We conclude that combinations of extracellular organisms can activate innate immune pathways previously considered to be reserved for the detection of intracellular microorganisms.