The One Health concept has proposed an integrated and unified approach aiming for health balance and enhancement by recognizing the interdependence of human, animal, and environmental health. The COVID-19 pandemic has pushed global One Health initiatives and policy improvement toward preventive measures for future pandemics, particularly of zoonotic origin. Such a scenario may be particularly relevant for South America, which is considered highly vulnerable due to its natural biodiversity superposed to socioeconomic and environmental issues, demanding effective methods and indicators for proper One Health strategies and goals that are aligned with macroregional contexts. Accordingly, the present study aimed to assess the One Health Index (OHI) in South American countries, along with potential interactions with socioeconomic indicators. The results obtained using clustering analysis and permutational multivariate analysis of variance (PERMANOVA) have revealed a positive association between the OHI and the Human Development Index (HDI) but not with gross domestic product (GDP). Although South American countries with political stability, robust investment in health, and progressive policies have shown a higher OHI, better environmental health is not associated with better human and animal health. In addition, although the Amazon biome- spanning 9 of the 12 South American countries-has positively impacted environmental health, this benefit contrasts with the rudimentary local human health systems, highlighting the complexity of One Health within the South American context. The lack of stronger indicators for animal health was also considered an important weak point for a true OHI assessment. Nonetheless, countries with more developed livestock have presented better animal health, which may not reflect an overall animal health indicator, as companion and wildlife animal health indicators were not available. Although lower (within-country) scale analysis such as states and metropolitan areas may better shape internal differences, the study herein has clearly shown One Health inequalities and challenges among South American countries. Equally important, forests and other natural areas in developing countries, particularly the Amazon, should receive incentives to promote sustainable economic growth. This approach would help prevent sacrificing environmental health for the benefit of human and livestock animal health.