Deciduous dipterocarp forests throughout Asia provide crucial habitat for several globally threatened species. During the dry season water availability in these forests is primarily limited to perennial rivers and waterholes. Such water sources form an essential part of these dry forests and are used by multiple species, including large mammals and birds, but little is known regarding how waterhole characteristics affect wildlife use. We investigated waterhole utilization by six globally threatened dry forest specialists: banteng Bos javanicus, Eld's deer Rucervus eldii, giant ibis Thaumatibis gigantea, green peafowl Pavo muticus, lesser adjutant Leptoptilos javanicus and Asian woolly-necked stork Ciconia episcopus. We camera-trapped 54 waterholes in Srepok Wildlife Sanctuary, eastern Cambodia, during the dry season of December 2015–June 2016. We measured nine waterhole and landscape characteristics, including indicators of human disturbance. Waterhole depth (measured every 2 weeks) and the area of water at the start of the dry season were the main environmental factors influencing waterhole use. Additionally, waterholes further from villages were more frequently used than those nearer. Our study reaffirmed the importance of waterholes in supporting globally threatened species, especially large grazers, which are critical for maintaining these dry forest ecosystems. The results also suggested that artificially enlarging and deepening selected waterholes, particularly those further from human disturbance, could enhance available habitat for a range of species, including grazers. However, this would need to be conducted in coordination with patrolling activities to ensure waterholes are not targets for illegal hunting, which is a problem throughout South-east Asian protected areas.