Poaching wildlife species and eradicating predators from game reserves are not considered crimes per se under Spanish legislation. Furthermore, in accordance with Spain?s Penal Code and Constitution, any doubt regarding a suspect?s guilt manda tes a judge/cr own prosecutor to rest a case without presenting charges. By contrast, the use of a substance with the intent to poison wildl ife is deemed both an offense according to the Wildlife Act (2003) and a crime under the Spanish Penal Code. Here we report on the atypical analysis of a unique piece of evidence that was retrieved during an on -site investigation into allegations of wildlife poaching and poisoning on a game reserve in southern Spain. The findings proved critical in linking other recovered evidence to the suspect and, in particular, showing intent to poison, which ultimately led to the closure of the case and the addition of another tool in the growing coffer of approaches for resolving similar cases in Andalucía. To our knowledge this is the first inst ance in which the presence of bacteria and evaluation of physico -chemical parameters have been used as integral pieces of evidence in a wildlife poisoning case.