Simple Summary: This research aims to better understand the causes of Mycosis fungoides (MF)—a disease where a type of immune cell, the T cell, malignantly transforms into cancer. It is not yet fully understood what triggers MF or how it progresses, partly because it varies so much between patients and because there is debate about whether the disease begins in immature T cells (thymocytes) or in more mature T cells (memory T cells). Recent findings suggest that bacteria living on the skin, particularly a harmful strain of Staphylococcus aureus, may aggravate MF by triggering specific pathways in T cells. To investigate this hypothesis, we explored the gene expression and microbial abundance of MF patients' skin with advanced statistical methods. We found that varied microbial skin colonization between patients may explain why the skin gene expression is so different from patient to patient. We also observed additional evidence that S. aureus might indeed trigger pathways in mature T cells that fuel cancer progression. Further, our statistical model suggested that certain viruses, like Epstein–Barr virus, could play a role in starting the disease by disrupting thymocytes (immature T cells). Based on these results, we speculate that both perspectives on the origin of MF could be correct, immature (thymocytes) and mature T cells: Thymocytes undergo malignant transformation, possibly caused by viruses, and bacteria like S. aureus fuel the malignant T cells to become prominent cancer cells. Our research could help uncover the complex interplay between bacteria, viruses, and T cells in MF. The findings may pave the way for new treatments targeting the skin microbiome or T cell pathways, offering hope for better management of this challenging disease. Background: Mycosis fungoides (MF) represents the most prevalent entity of cutaneous T cell lymphoma (CTCL). The MF aetiopathogenesis is incompletely understood, due to significant transcriptomic heterogeneity and conflicting views on whether oncologic transformation originates in early thymocytes or mature effector memory T cells. Recently, using clinical specimens, our group showed that the skin microbiome aggravates disease course, mainly driven by an outgrowing, pathogenic S. aureus strain carrying the virulence factor spa, which was shown by others to activate the T cell signalling pathway NF-κB. Methods: To explore the role of the skin microbiome in MF aetiopathogenesis, we here performed RNA sequencing, multi-omic data integration of the skin microbiome and skin transcriptome using Multi-Omic Factor Analysis (MOFA), virome profiling, and T cell receptor (TCR) sequencing in 10 MF patients from our previous study group. Results: We observed that inter-patient transcriptional heterogeneity may be largely attributed to differential activation of T cell signalling pathways. Notably, the MOFA model resolved the heterogenous activation pattern of T cell signalling after denoising the transcriptome from microbial influence. The MOFA model suggested that the outgrowing S. aureus strain evoked signalling by non-canonical NF-κB and IL-1B, which in turn may have fuelled the aggravated disease course. Further, the MOFA model indicated aberrant pathways of early thymopoiesis alongside enrichment of antiviral innate immunity. In line with this, viral prevalence, particularly of Epstein–Barr virus (EBV), trended higher in both lesional skin and the blood compared to nonlesional skin. Additionally, TCRs in both MF skin lesions and the blood were significantly more likely to recognize EBV peptides involved in latent infection. Conclusions: First, our findings suggest that S. aureus with its virulence factor spa fuels MF progression through non-canonical NF-κB and IL-1B signalling. Second, our data provide insights into the potential role of viruses in MF aetiology. Last, we propose a model of microbiome-driven MF aetiopathogenesis: Thymocytes undergo initial oncologic transformation, potentially caused by viruses. After maturation and skin infiltration, an outgrowing, pathogenic S. aureus strain evokes activation and maturation into effector memory T cells, resulting in aggressive disease. Further studies are warranted to verify and extend our data, which are based on computational analyses. [ABSTRACT FROM AUTHOR]