Southern Afrotemperate Forest is concentrated in the southern Cape region of South Africa and whilst it is relatively well known botanically, the fauna, specifically the aquatic invertebrate fauna, is poorly documented. The majority of remaining intact forest habitat is contained within the Garden Route National Park (GRNP), which straddles the provincial boundary between the Western and Eastern Cape. This study undertakes a survey of the water beetle fauna inhabiting the GRNP. The aquatic ecosystems within temperate forests of the region are poorly researched from an ecological and biodiversity perspective, despite being known to harbour endemic invertebrate elements. We collected water beetles and in situ physico-chemical data from a total of 31 waterbodies across the park over two seasons (summer and late winter) in 2017. The waterbodies sampled were mostly small freshwater perennial streams and isolated forest ponds. A total of 61 beetle taxa was recorded (29 Adephaga, 32 Polyphaga) from these waterbodies. The water beetle fauna of these forests appears to be diverse and contains many species endemic to the fynbos-dominated Cape Floristic Region, but very few of the species appear to be forest specialists. This is in contrast to the fynbos heathland habitat of the region, which harbours a high number of water beetle species endemic to this habitat, often with Gondwanan affinity. Our study is the first to document the water beetles of Afrotemperate Forests in the southern Cape region and provides an important baseline for future work on such habitats in the region and in other parts of southern Africa.