The upper Neretva River valley with river and tributaries, preserved forests, natural caves and buildings represents a very interesting area for bats. During two field expeditions in summer 2022, we collected data on bats using a variety of methods: inspecting the potential roosts, mist netting, and recording bat echolocation calls. We used manual and automatic ultrasound detectors most frequently at sites along and near the Neretva River. The morphological identification of some species was confirmed using DNA analyses. We detected at least 13 bat species in the study area extending from Krupac to Konjic. With one addition, known from the literature, 14 different bat species have been found in the upper Neretva River valley, presenting nearly a half of all bat species recorded in Bosnia and Herzegovina. We confirmed the presence of three species of the highest conservation concern in Europe, listed in the Annex II of the Habitats directive: lesser horseshoe bat (Rhinolophus hipposideros), greater horseshoe bat (Rhinolophus ferrumequinum), and greater mouse-eared bat (Myotis myotis). Our finding of the alcathoe bat (Myotis alcathoe) presents new confirmation of this species in Bosnia and Herzegovina. Also, observation of the parti-coloured bat (Vespertilio murinus) is one of the few records of the species in this country. The upper Neretva River valley can be considered as an area of high conservation importance for bats. Additional fieldwork, including during other seasons, would likely reveal more bat species and their sites in the area of the upper Neretva River valley.