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Sphaerellopsisspecies are putative hyperparasites of rust fungi and may be promising biological control agents (BCA) of rust diseases. However, few detailed studies limit potential BCA development in Sphaerellopsis. Here, we explored the biogeography, host-specificity, and species diversity of Sphaerellopsisand examined the early infection stage of one species, S. macroconidialis,to infer its trophic status. We randomly screened 5,621 rust specimens spanning 99 genera at the Arthur Fungarium for the presence of Sphaerellopsis. We identified 199 rust specimens infected with Sphaerellopsisspecies on which we conducted morphological and multi-locus phylogenetic analyses. Five Sphaerellopsisspecies were recovered, infecting a total of 122 rust species in 18 genera from 34 countries. Sphaerellopsis melampsorinearumsp. nov. is described as a new species based on molecular phylogenetic data and morphological features of the sexual and asexual morphs. Sphaerellopsis paraphysatawas the most commonly encountered species, found on 77 rust specimens, followed by Sphaerellopsis macroconidialison 56 and S. melampsorinearumon 55 examined specimens. The type species, Sphaerellopsis filum, was found on 12 rust specimens and Sphaerellopsis hakeaeon a single specimen. We also recovered and documented for the first time, the sexual morph of S. macroconidialis, from a specimen collected in Brazil. Our data indicate that Sphaerellopsisspecies are not host specific and furthermore that most species are cosmopolitan in distribution. However, S. paraphysatais more abundant in the tropics, and S. hakeaemay be restricted to Australia. Finally, we confirm the mycoparasitic strategy of S. macroconidialisthrough in-vitrointeraction tests with the urediniospores of Puccinia polysora. Shortly after germination, hyphae of S. macroconidialisbegan growing along the germ tubes of P. polysoraand coiling around them. After 12 days of co-cultivation, turgor loss was evident in the germ tubes of P. polysora, and appressorium-like structures had formed on urediniospores. The interaction studies indicate that Sphaerellopsisspecies may be more effective as a BCA during the initial stages of rust establishment.