Illudin S (ILS) is a fungal sesquiterpene secondary metabolite with potent genotoxic and cytotoxic properties. Early genetic studies and more recent genome-wide CRISPR screens showed that Illudin-induced lesions are preferentially repaired by transcription-coupled nucleotide excision repair (TC-NER) with some contribution from post-replication repair pathways. In line with these results, Irofulven, a semi-synthetic ILS analog was recently shown to be particularly effective on cell lines and patient-derived xenografts with impaired NER (e.g. ERCC2/3 mutations), raising hope that ILS-derived molecules may soon enter the clinic. Despite the therapeutic potential of ILS and its analogs, we still lack a global understanding of their mutagenic potential. Here, we characterize the mutational signatures associated with chronic exposure to ILS in human cells. ILS treatment rapidly stalls DNA replication and transcription, leading to the activation of the replication stress response and the accumulation of DNA damage. Novel single and double base substitution signatures as well as a characteristic indel signature indicate that ILS treatment preferentially alkylates purine residues and induces oxidative stress, confirming prior in vitro data. Many mutation contexts exhibit a strong transcriptional strand bias, highlighting the contribution of TC-NER to the repair of ILS lesions. Finally, collateral mutations are also observed in response to ILS, suggesting a contribution of translesion synthesis pathways to ILS tolerance. Accordingly, ILS treatment led to the rapid recruitment of the Y-family DNA polymerase kappa onto chromatin, supporting its preferential use for ILS lesion bypass. Altogether, our work provides the first global assessment of the genomic impact of ILS, demonstrating the contribution of multiple DNA repair pathways to ILS resistance and mutagenicity.