Semuliki National Park is located in Bwamba County, a remote part of the Bundibugyo District in western Uganda. Semuliki National Park has an area of 219 sq km and is part of the Central African Congo Basin forest system of the Democratic Republic of Congo (DRC), being separated from the Ituri forest of the DRC only by the Semliki River. It is separated from the rest of East Africa by the Rwenzori Mountain range and with it being located within the Albertine Rift, the western arm of the Great Rift Valley, it is included within the Eastern Afromontane biodiversity hotspot
The park experiences an average rainfall of 1,250 mm, with peaks in rainfall from March to May and from September to December. Many areas of the park experience flooding during the wet season. The temperature at the park varies from 18 to 30 °C, with relatively small daily variations.
The area that Semuliki National Park covers is a distinct ecosystem within the larger Albertine Rift ecosystem. The park is located at the junction of several climatic and ecological zones, and as a result has a high diversity of plant and animal species and many microhabitats. Most of the plant and animal species in the park are also found in the Congo Basin forests, with many of these species reaching the eastern limit of their range in Semuliki National Park.