The freshwater ecosystems around the world are degrading, such that maintaining environmental flow (EF) in river networks is critical to their preservation. The relationship between streamflow alterations and, respectively, EF violations, and freshwater biodiversity is well established at the scale of stream reaches or small basins (~<100 km²). However, it is unclear if this relationship is robust at larger scales even though there are large-scale initiatives to legalize the EF requirement. Moreover, EFs have been used in assessing a planetary boundary for freshwater. Therefore, this study intends to carry out an exploratory evaluation of the relationship between EF violation and freshwater biodiversity at globally aggregated scales and for freshwater ecoregions. Four EF violation indices (severity, frequency, the probability to shift to violated state, and probability to stay violated) and seven independent freshwater biodiversity indicators (calculated from observed biota data) were used for correlation analysis. No statistically significant negative relationship between EF violation and freshwater biodiversity was found at global or ecoregion scales. While our results thus suggest that streamflow and EF may not be an only determinant of freshwater biodiversity at large scales, they do not preclude the existence of relationships at smaller scales or with more holistic EF methods (e.g., including water temperature, water quality, intermittency, connectivity etc.) or with other biodiversity data or metrics.