Iron and sulfur are indispensable elements of every living cell, but on their own these elements are toxic and require dedicated machineries for the formation of iron/sulfur (Fe/S) clusters. In eukaryotes, proteins requiring Fe/S clusters (Fe/S proteins) are found in or associated with various organelles including the mitochondrion, endoplasmic reticulum, cytosol, and the nucleus. These proteins are involved in several pathways indispensable for the viability of each living cell including DNA maintenance, protein translation and metabolic pathways. Thus, the formation of Fe/S clusters and their delivery to these proteins has a fundamental role in the functions and the evolution of the eukaryotic cell. Currently, most eukaryotes harbor two (located in cytosol and mitochondrion) or three (located in plastid) machineries for the assembly of Fe/S clusters, but certain anaerobic microbial eukaryotes contain sulfur mobilization (SUF) machineries that were previously thought to be present only in archaeal linages. These machineries could not only stipulate which pathway was present in the last eukaryotic common ancestor (LECA), but they could also provide clues regarding presence of an Fe/S cluster machinery in the proto-eukaryote and evolution of Fe/S cluster assembly machineries in all eukaryotes.