Merkel cell polyomavirus (MCPyV) has been detected in respiratory specimens including those from Cystic Fibrosis (CF) patients, raising questions about its immunological and clinical relevance in the respiratory tract. MCPyV is considered the etiological agent of Merkel cell carcinoma (MCC) and, considering the widespread prevalence of the virus across the body, the involvement of MCPyV in tumours other than MCC cannot be excluded. It has been proposed that MCPyV could be associated with non-MCC cancers such as non-small-cell lung carcinoma (NSCLC). A potential association between MCPyV infection and EGFR expression it has also been suggested since BRAF gene, a downstream target of EGFR pathway, was found to be higher expressed in MCPyV positive samples than negative ones. In this study, as objective one, the prevalence of MCPyV-DNA in respiratory samples of a large cohort of CF patients was estimated analyzing viral load and sequencing the LT, NCCR and VP1 regions. In addition, in order to shed light on the potential pathogenic role of MCPyV in CF, demographic, microbiological and clinical data collected from MCPyV-DNA positive and negative patients were compared. Moreover, the transcript expression of TLR9 and distinct IFN-I genes (IFNα, IFNβ and IFNε) were also examined in respiratory samples of MCPyV positive CF patients according to their bacteriological and clinical status. Respiratory samples (n = 1138) were randomly collected from respiratory tract of CF patients (n = 539). MCPyV-DNA was detected in 268 out of 1138 respiratory specimens (23.5%), without any difference in the prevalence of MCPyV-DNA, according to age, gender or bacteriological status of CF individuals. Thirteen out of 137 CF patients remained positive for MCPyV-DNA over the time. Detection of MCPyV-DNA in respiratory specimens was not associated with the occurrence of exacerbation events. Both MCPyV positive adolescents (11–24 years) and adults (≥25 years) had lower mRNA levels of TLR9, IFNβ, IFNε and IFNα than the negative patients of the same age group, while MCPyV positive children produced increased levels of TLR9 and IFN-I genes (p < 0.05 for TLR9, IFNβ, IFNε) with respect to the negative ones. There were significant differences in TLR9 levels (p < 0.01), but not in those of IFNs, between MCPyV-DNA positive and negative patients with S. aureus, P. aeruginosa or both. Overall, these results indicate that MCPyV-DNA is frequently detected in the respiratory samples of CF patients and might influence the expression levels of IFN-related genes in an age dependent manner. The concomitant detection of MCPyV together with S. aureus and/or P. aeruginosa correlated with alterations in TLR9 levels suggesting that virus-bacteria coinfections might contribute to affect antiviral immunity in CF patients. The second aim of this study was to investigate the role of MCPyV as etiological viral agent of NSCLCs by examining a series of NSCLC-patients for both the presence of specific MCPyV DNA and the expressions of viral RNA transcripts (LT and VP1). The integrated form of the MCPyV-positive NSCLCs was also examinate. Formalin-fixed paraffin-embedded tissue (FFPE) of NSCLCs and corresponding nonmalignant lung tissue were obtained from 112 Italian patients undergoing surgery. PCR revealed that 9 out of 32 squamous cell carcinomas (SCCs), 9 out of 45 adenocarcinomas, 1 out of 32 large-cell carcinomas, and 1 out of 3 pleomorphic carcinomas were positive for MCPyV DNA. Some MCPyV DNA-positive cancers expressed LT RNA transcripts. The viral integration sites were identified in one SCC. The other samples didn’t show an integrated MCPyV genome but frameshift mutations in the LT gene. Analysis of EGFR showed that the infection rate of MCPyV was higher in NSCLCs with EGFR mutations than without EGFR mutations; however, this difference was not statistically significant. The obtained results showed the expression of a viral oncoprotein, the presence of integrated MCPyV, and a truncated LT gene with a preserved retinoblastoma tumor-suppressor protein-binding domain in NSCLCs. Although the viral prevalence was low, the tumor-specific molecular signatures support the possibility that MCPyV could be associated with the pathogenesis of NSCLC in a subset of patients. To conclude, since MCPyV infection is observed in occurrence of EGFR mutation, our results could indicate that MCPyV could be considered an EGFR mutagen and might spark interests to deepen the prognostic value of EGFR in NSCLCs.