Gambusia holbrooki is one of the most problematic invasive species globally, but its presence has yet to be confirmed in some regions. This is the case of the particularly conservation-sensitive freshwater insular ecosystems of Madeira Archipelago in Macaronesia. Freshwater ecosystems on the two main islands were inspected based on previous unconfirmed observations of mosquitofish. Specimens were found in three artificial ponds in Madeira Island and in one stream of Porto Santo Island. The morphology and mitochondrial DNA of the collected specimens confirmed their identification as G. holbrooki, confirming its presence on this archipelago for the first time. However, previous records suggest that the species may have inhabited Madeira Island for decades. Also, the high number of individuals detected in Porto Santo suggests an established population on this island. The apparent population size in Porto Santo may make an eradication action extremely challenging. Nonetheless, this may still be achievable on Madeira Island, which is extensively covered by inland waters. Proactive measures are essential to prevent further spread and mitigate potential biodiversity loss in freshwater ecosystems of this archipelago. Measures must be adapted to account for the current mosquitofish distribution and the reduced carrying capacity of Madeira’s freshwater ecosystems. It is also important to consider that these ecosystems serve as habitat for an endangered native fish species and several endemic macroinvertebrates. Ultimately, it is essential to develop a monitoring plan to avoid introductions into adjacent natural areas and towards other insular ecosystems.