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High-throughput RNA sequencing (RNA-seq) is routinely applied to study diverse biological processes; however, when performed separately on interacting organisms, systemic noise intrinsic to RNA extraction, library preparation, and sequencing hampers the identification of cross-species interaction nodes. Here, we develop triple RNA-seq to simultaneously detect transcriptomes of monocyte-derived dendritic cells (moDCs) infected with the frequently co-occurring pulmonary pathogens Aspergillus fumigatus and human cytomegalovirus (CMV). Comparing expression patterns after co-infection with those after single infections, our data reveal synergistic effects and mutual interferences between host responses to the two pathogens. For example, CMV attenuates the fungus-mediated activation of pro-inflammatory cytokines through NF-κB (nuclear factor κB) and NFAT (nuclear factor of activated T cells) cascades, while A. fumigatus impairs viral clearance by counteracting viral nucleic acid-induced activation of type I interferon signaling. Together, the analytical power of triple RNA-seq proposes molecular hubs in the differential moDC response to fungal/viral single infection or co-infection that contribute to our understanding of the etiology and, potentially, clearance of post-transplant infections. • Triple RNA-seq measures gene expression of co-infected immune cells • Gene correlation networks reveal different hub gene sets under co-infection • Co-infection expression includes synergies and interferences between host and pathogens • Molecular basis of viral/fungal pulmonary infection has potential for the clinic Seelbinder et al. demonstrate simultaneous sequencing of RNA isolated from human immune cells infected with Aspergillus fumigatus and cytomegalovirus, two pulmonary pathogens regularly affecting the lungs of immunosuppressed patients. They detect characteristic gene expression patterns for single and co-infections and reveal synergistic virulence strategies between the two pathogens. [ABSTRACT FROM AUTHOR]