Neglected tropical zoonotic diseases (NTZDs) continue to have a major effect on the health of humans and animals. In this study, a one health approach was used to prioritize and rank neglected tropical zoonotic diseases at the regional and zonal levels in Tigray National Regional State, Ethiopia. For prioritization of NTZDs a cross-sectional study through a structured questionnaire was administered to 313 health experts from human and animal health sectors. In addition, focus group discussions (FGD) were held with purposively selected key informants. Descriptive, and Multivariable analysis was applied to report the results and a ranked list of diseases was developed at the zonal and regional level. In the region, 8 of the 12 World Health Organization listed NTZDs were considered major diseases including anthrax, brucellosis, bovine tuberculosis, taeniasis, leishmaniasis, rabies, schistosomiasis, and soil-transmitted helminths. Considering the zoonotic and socioeconomic importance of the diseases at the regional level, rabies ranked 1stwhereas anthrax, bovine tuberculosis, leishmaniasis, and brucellosis were ranked from 2nd to 5th, respectively. The FGD result also supported the prioritization result. The Multivariable analysis showed a statistically significant difference in the zonal distribution of anthrax (р = 0.009, OR = 1.16), taeniasis (p<0.001, OR = 0.82), leishmaniasis (p<0.001, OR = 1.91), rabies (p = 0.020, OR = 0.79) and soil-transmitted helminths (p = 0.007, OR = 0.87) but not for brucellosis (p = 0.585), bovine tuberculosis (p = 0.505), and schistosomiasis (p = 0.421). Anthrax (p<0.001, OR = 26.68), brucellosis (p<0.001, OR = 13.18), and taeniasis (p<0.001, OR = 6.17) were considered as the major zoonotic diseases by veterinary practitioners than human health practitioners whereas, leishmaniasis was recognized as a major health challenge by human health professionals. Understanding the priority diseases in the region is supportive for informed decision-making and prioritizes the limited resources to use. Furthermore, strengthening the collaboration between human and animal health professions is important to control the diseases.