The 2020 mass mortality of 350 African elephants (Loxodonta africana) in Botswana sparked global concern, with cyanotoxins in watering holes (pans) being a suspected cause, though evidence remains inconclusive. Combining remote sensing and spatial analysis, we examined the relationship between the ecohydrology of ∼3000 pans and the locations of deceased elephants. Our analysis revealed a significant difference in the spatial distribution of fresh versus decayed carcasses (p < 0.001), indicating that the die-off deviated from typical regional elephant mortality patterns. We identified 20 pans near fresh carcasses that experienced increased cyanobacteria bloom events in 2020 (n = 123) compared to the previous 3 years combined (n = 23), exhibiting the highest average phytoplankton biomass of the period 2015–2023 (Normalised Difference Chlorophyll Index >0.2). These findings suggest a heightened risk and likelihood of cyanotoxin presence in these pans. Elephants were estimated to have walked 16.5 km (± 6.2 km) and died within 88 h (± 33 h) of exposure. Our study provides evidence that cyanobacterial toxicity could be a contributing factor to the 2020 die-off, while also considering other potential causes, and offers a general framework for investigation of future mortality events. We underscore the need to integrate spatial analysis and regional ecohydrological assessments to monitor and mitigate animal mortality events and inform conservation strategies.