<p>A vast portion of genes in microbial genomes, termed the “unknome,” remains functionally uncharacterised. This genetic “dark matter” represents a significant bottleneck in microbiology, as it is often excluded from genomic studies. We argue that a substantial part of the unknome encodes functions critical for biotic interactions, the complex dialogues among microbes or between microbes and their hosts. These functions are rarely observed under standard laboratory conditions, which rely on simplified pure cultures. Unlocking the unknome therefore calls for a stronger emphasis on ecologically relevant experimental systems. By embracing complexity through co-culture and in situ analyses, we can begin to decipher this hidden genetic repertoire, deepening our understanding of microbial communication, adaptation, and evolution. Crucially, the conceptual and methodological challenges raised by the microbial unknome resonate well beyond microbiology: parallel “dark” fractions of uncharacterised genes and proteins pervade eukaryotic genomes, from lineage-specific (orphan) genes underpinning novelties in plants and animals, including humans. Embracing ecological and systems-level approaches to dissect the unknome therefore has the potential to reframe how we link genotype to phenotype in context-dependent, interaction-driven biological systems.</p>