Fifty years ago, Lynn Margulis, inspiring in early twentieth-century ideas that put forward a symbiotic origin for some eukaryotic organelles, proposed a unified theory for the origin of the eukaryotic cell based on symbiosis as evolutionary mechanism. Margulis was profoundly aware of the importance of symbiosis in the natural microbial world and anticipated the evolutionary significance that integrated cooperative interactions might have as mechanism to increase cellular complexity. Today, we have started fully appreciating the vast extent of microbial diversity and the importance of syntrophic metabolic cooperation in natural ecosystems, especially in sediments and microbial mats. Also, not only the symbiogenetic origin of mitochondria and chloroplasts has been clearly demonstrated, but improvement in phylogenomic methods combined with recent discoveries of archaeal lineages more closely related to eukaryotes further support the symbiogenetic origin of the eukaryotic cell. Margulis left us in legacy the idea of ‘eukaryogenesis by symbiogenesis’. Although this has been largely verified, when, where, and specifically how eukaryotic cells evolved are yet unclear. Here, we shortly review current knowledge about symbiotic interactions in the microbial world and their evolutionary impact, the status of eukaryogenetic models and the current challenges and perspectives ahead to reconstruct the evolutionary path to eukaryotes.