Evolution of virulence in parasites has profound effects on both host-parasite co-evolution and ecology and is influenced by environmental factors and the genotypes involved. Many parasite infections consist of multiple strains or species that are predicted to result in the evolution of more virulent strains that exploit the host less prudently. In opportunistic parasites, the dynamics and evolution of virulence are poorly understood as traditional epidemiological models do not adequately describe parasites capable of persisting outside of the host. In addition, as microbial pathogenicity may shift from opportunistic to obligatory strategies with time, knowledge of the evolutionary dynamics of opportunistic pathogens is crucial for predicting and understanding disease emergence. Aspergillus species of fungi have a ubiquitous distribution and are the etiological agents of stonebrood disease in honey bees. It is generally considered that stonebrood occurs rarely in honey bees, but the epidemiology and predisposing conditions for the disease are almost entirely unknown. In this study, I examine the occurrence, pathogenicity and competitive ability of Aspergillus spp. when infecting honey bees, as well as the effect of nutrition on host susceptibility and the adaptation of the fungi over the course of experimental evolution. A high prevalence and diversity of Aspergillus spp. isolates were identified following the screening of an apiary, and the pathogenicity of three species (A. flavus, A. nomius and A. phoenicis) was established. Further, in laboratory-reared larvae a nutritionally limited diet increased susceptibility to A. fumigatus. In a series of single-generation interspecific competition experiments between Aspergillus spp. and the obligate pathogen chalkbrood (Ascophaera apis), the virulence and fitness of dual infections were influenced by complex within-host interactions depending on the species involved, which ranged from synergistic to inhibitory effects. Finally, following serial passage of A. flavus and A. phoenicis in the honey bee larval hosts to determine the evolution of virulence and fitness, no evidence of host adaptation was observed, revealing the unpredictability of these asexually reproducing opportunistic pathogens. These findings illustrate a complex relationship between Aspergillus spp. and honey bees and emphasises the significant influence these ubiquitous organisms can have on the ecology and evolution of honey bees.