Background The frst 24 h of infection represent a critical time window in interactions between pathogens and host tissue. However, it is not possible to study such early events in human lung during natural infection due to lack of clinical access to tissue this early in infection. We, therefore, applied RNA sequencing to ex vivo cultured human lung tissue explants (HLTE) from patients with emphysema to study global changes in small noncoding RNA, mRNA, and long noncoding RNA (lncRNA, lincRNA) populations during the frst 24 h of infection with infuenza A virus (IAV), Mycobacterium bovis Bacille Calmette-Guerin (BCG), and Pseudomonas aeruginosa. Results Pseudomonas aeruginosa caused the strongest expression changes and was the only pathogen that notably afected expression of microRNA and PIWI-associated RNA. The major classes of long RNAs (>100 nt) were represented similarly among the RNAs that were diferentially expressed upon infection with the three pathogens (mRNA 77–82%; lncRNA 15–17%; pseudogenes 4–5%), but lnc-DDX60-1, RP11-202G18.1, and lnc-THOC3-2 were part of an RNA signature (additionally containing SNX10 and SLC8A1) specifcally associated with IAV infection. IAV infection induced brisk interferon responses, CCL8 being the most strongly upregulated mRNA. Single-cell RNA sequencing identifed airway epithelial cells and macrophages as the predominant IAV host cells, but infammatory responses were also detected in cell types expressing few or no IAV transcripts. Combined analysis of bulk and single-cell RNAseq data identifed a set of 6 mRNAs (IFI6, IFI44L, IRF7, ISG15, MX1, MX2) as the core transcriptomic response to IAV infection. The two bacterial pathogens induced qualitatively very similar changes in mRNA expression and predicted signaling pathways, but the magnitude of change was greater in P. aeruginosa infection. Upregulation of GJB2, VNN1, DUSP4, SerpinB7, and IL10, and downregulation of PKMYT1, S100A4, GGTA1P, and SLC22A31 were most strongly associated with bacterial infection. Conclusions Human lung tissue mounted substantially diferent transcriptomic responses to infection by IAV than by BCG and P. aeruginosa, whereas responses to these two divergent bacterial pathogens were surprisingly similar. This HLTE model should prove useful for RNA-directed pathogenesis research and tissue biomarker discovery during the early phase of infections, both at the tissue and single-cell level. Keywords Biomarker, Chronic obstructive pulmonary disease, Emphysema, Gene expression, Lung tissue, Infection, Long noncoding RNA, Pneumonia, miRNA, PIWI-associated RNA, Prokineticin 2, Transcription