Throughout the history of molecular biology, surprising advances have come from the study of all sorts of microbes. The first description of DNA polymerase came from the bacterial workhorse Escherichia coli, reverse transcriptase was revealed by studies of the Rous Sarcoma Virus, and even the initial discovery of DNA as the hereditary molecule was determined using strains of pneumococci in the classic Griffith and Avery–MacLeod–McCarty experiments. Here, we build from these foundational discoveries to discuss the rapid development of molecular tools to study microbes themselves, with a focus on nucleic acid biology. We use fungal pathogens as a case study, as their diversity, complexity, and emerging appreciation as a global threat to society makes them particularly compelling and informative. In this review, we will address how advancements in methods to probe nucleic acids are now informing our understanding of fungal pathogens and the way we fight them in both the clinic and agriculture. We begin with DNA, taking a close look at the exciting progress in the fields of genetic engineering and chromatin biology, and their impact on the elucidation of virulence-associated cellular processes. Emerging RNA-based technologies follow, highlighting the value provided by biochemical advances and large-scale -omics approaches. We end by speculating on the future of molecular mycology and how these new approaches may facilitate generation of novel antifungals and diagnostic strategies.