<p>Rust fungi (Pucciniales) are plant pathogens that cause diseases on economically important crops worldwide and threaten native plants with extinction. Fungicides and disease-resistant plant varieties are the two primary options to control rust diseases. However, more aggressive rust races are emerging, and the current control methods are not as efficient at managing disease as they have been in the past. Thus, we must find other alternatives to keep rust fungi at low population levels to prevent high-yield losses and preserve our natural ecosystems. Biological control (BC) is one promising alternative to the current control methods. BC uses natural enemies (NEs), such as insects and microorganisms, to control pests and plant diseases. However, because the NEs of rust fungi are scarcely documented and not well characterized, using BC for rust diseases is not feasible with the current state of knowledge. Characterization studies of NEs of rust fungi are essential to select or discard potential biological control agents. How do NEs infect or feed the rust? Are NEs specific to rust species or genera? How diverse are they? and how adapted are they to different geographic regions? These are a few of the unresolved questions needed to answer to tap the potential of NEs in applied research for the biological control of rust diseases. This dissertation thesis had three aims: 1) to catalog the NEs of rust fungi from the Arthur Fungarium (PUR) rust collections emphasizing specimens from the Americas and providing barcoding sequences, photographs, and morphological descriptions. This was accomplished by thoroughly screening ca. 50% of the accepted rust genera collected over 200 years from across the globe and from 166 host plant families; 2) to describe new fungal species associated with rust fungi; and 3) to characterize the two most common NEs of rust fungi in aspects related to species diversity, host preference, geographic distribution, and antagonistic interactions. Of the 5,618 randomly screened rust specimens, we found 543 specimens with fungal NEs or hyperparasites. These belonged to the fungal genera <em>Helicobasidium</em>, <em>Ramularia</em>, <em>Sphaerellopsis</em>, <em>Simplicillium</em>, and <em>Trochila</em>. In addition to fungal antagonists, we found larvae of the gall midge <em>Mycodiplosis</em>, whose larval stage feeds rust spores, present in 287 specimens. Among these NEs, the fungal genus <em>Sphaerellopsis</em> and the fly larvae <em>Mycodiplosis</em> were the most commonly associated with rust fungi samples. Therefore, we focused on members of these two genera to determine their host-specificity, geographic distribution and describe early antagonistic interaction events. Our results suggest that members of <em>Sphaerellopsis</em> and <em>Mycodiplosis</em> do not prefer specific rust species or genera. Thus, further studies on non-target species are needed to determine how broad their host range is. In addition, we found that both NEs have a cosmopolitan distribution. However, the fly larvae of <em>Mycodiplosis</em> show distribution patterns, suggesting that some of these species are regionally restricted, especially in the Americas. This result is an exciting finding for conservation biological control where native NEs could be stimulated to increase their populations and control targeted pests. Thus, <em>Mycodiplosis</em> larvae may be a good candidate for local use to control rust diseases without introducing non-native invasive species to new environments. Lastly, we describe the antagonistic interaction events between <em>Sphaerellopsis macroconidialis</em> and the urediniospores of southern corn rust, <em>Puccinia polysora</em>. Although <em>S. macroconidialis </em>was reported as a NE of rust fungi, the nature of the interaction had not been previously described. Herein, we observed how the hyphae of <em>S. macroconidialis</em> grew along the urediniospores germinative tube and tightly coiled around this structure. We also observed a turgor loss of the germinative tube a few days after interacting with the NE. Overall, these results contribute significantly to the characterization of the NEs of rust fungi for further studies in biological control and to develop sustainable agronomical practices in controlling rust diseases.</p>