Cut branches of deciduous holly (Ilex spp. L.) harboring colorful berries are traditionally used as ornaments in holiday decorations. Since 2012, a fruit rot of unspecified cause has resulted in significant yield reduction and economic losses across Midwestern and Eastern U.S. nurseries. In this study, symptomatic fruit samples collected from nine different locations over five years were analyzed, and several fungal species were isolated. A combination of morphological characterization, multilocus phylogenetic analyses, and pathogenicity assays revealed that Alternaria alternata and Diaporthe ilicicola sp. nov. were the primary pathogens associated with symptomatic fruit. Other fungi including A. arborescens, Colletotrichum fioriniae, C. nymphaeae, Epicoccum nigrum, and species in the D. eres species complex appeared to be minor pathogens in this disease complex. In detached fruit pathogenicity assays testing the role of wounding and inoculum concentration on disease development, disease incidence and severity increased when fruit was wounded and inoculated with a higher inoculum concentration. These findings indicate that management strategies that can protect fruit from injury or reduce inoculum may lower disease levels in the field. This research established the basis for further studies on this emerging disease and the design of research-based management strategies. To our knowledge, it also represents the first report of species of Alternaria, Colletotrichum, Diaporthe, and Epicoccum causing fruit rot of deciduous holly.