Iron-sulfur (Fe/S) cluster enzymes are crucial to life. Their assembly requires a suite of proteins, some of which are specific for particular subsets of Fe/S enzymes. One such protein is yeast Iba57p, which aconitase and certain radical S-adenosylmethionine enzymes require for activity. Iba57p homologs occur in all domains of life; they belong to the COG0354 protein family and are structurally similar to various folate-dependent enzymes. We therefore investigated the possible relationship between folates and Fe/S cluster enzymes using the Escherichia coli Iba57p homolog, YgfZ. NMR analysis confirmed that purified YgfZ showed stereoselective folate binding. Inactivating ygfZ reduced the activities of the Fe/S tRNA modification enzyme MiaB and certain other Fe/S enzymes, although not aconitase. When successive steps in folate biosynthesis were ablated, folE (lacking pterins and folates) and folP (lacking folates) mutants mimicked the ygfZ mutant in having low MiaB activities, whereas folE thyA mutants supplemented with 5-formyltetrahydrofolate (lacking pterins and depleted in dihydrofolate) and gcvP glyA mutants (lacking one-carbon tetrahydrofolates) had intermediate MiaB activities. These data indicate that YgfZ requires a folate, most probably tetrahydrofolate. Importantly, the ygfZ mutant was hypersensitive to oxidative stress and grew poorly on minimal media. COG0354 genes of bacterial, archaeal, fungal, protistan, animal, or plant origin complemented one or both of these growth phenotypes as well as the MiaB activity phenotype. Comparative genomic analysis indicated widespread functional associations between COG0354 proteins and Fe/S cluster metabolism. Thus COG0354 proteins have an ancient, conserved, folate-dependent function in the activity of certain Fe/S cluster enzymes.