New World vultures, such as turkey vultures (Cathartes aura), are obligate scavengers with large geographic ranges. In a preliminary characterization of the turkey vulture (TV) gastrointestinal microbiome in Southern California, we identified 2 recently described emerging bacterial pathogens not previously known to be associated with this avian species. High-throughput sequencing of broad-range 16S rRNA gene amplicons revealed sequences from TV cloacal swabs that were related closest to Wohlfahrtiimonas chitiniclastica and Ignatzschineria species, both Gammaproteobacteria considered by the United States Centers for Disease Control and Prevention as emerging zoonotic pathogens. None of these bacterial sequence types have been previously identified from samples obtained from the turkey vulture gastrointestinal microbiome. With the use of bioinformatics workflows previously established by our research group, we designed specific and sensitive polymerase chain reaction primer sets that represent novel diagnostic assays for the genera Wohlfahrtiimonas and Ignatzschineria. These primer sets were validated by Sanger sequence confirmation from complex TV samples. Because the genera Wohlfahrtiimonas and Ignatzschineria are both known to have dipteran hosts, the molecular diagnostic tools we present here should be useful for better understanding the role of flies, vultures, and other scavengers in the ecology and epidemiology of the genera Wohlfahrtiimonas and Ignatzschineria from a One Health perspective.