[7][8] Like the flora, the fauna of Thung Yai provides a specific mix of species with Sundaic, Indo-Chinese, Indo-Burmese and Sino-Himalayan affinities due to the sanctuary's particular biogeographic location.

[9] Among the mammal species living in Thung Yai are lar gibbon (Hylobates lar), various species of macaque (Macaca) and lutung (Trachypithecus), Indochinese tiger (Panthera tigris tigris), Indochinese leopard (Panthera pardus delacouri), clouded leopard (Neofelis nebulosa), sun bear (Helarctos malayanus) and Asian black bear (Ursus thibetanus), Malayan tapir (Tapirus indicus), Indian elephant (Elephas maximus indicus), gaur (Bos gaurus), hog deer (Cervus porcinus), sambar (Rusa unicolor), Fea's muntjac (Muntiacus feae) und Sumatran serow (Capricornis sumatraensis) as well as many bat species probably including Kitti's hog-nosed bat (Craseonycteris thonglongyai).

Thung Yai is part of the Western Forest Complex, which is the largest tiger habitat in the Southeast Asia region, with around 200 of the animals living there.

[9] Banteng (Bos javanicus) and wild water buffalo (Bubalus amee) are known to occur in the adjoining Huai Kha Khaeng Wildlife Sanctuary and may exist in Thung Yai too.

[7] Bird species sighted in Thung Yai include white-winged wood duck (Cairina scutulata), kalij pheasant (Lophura leucomelanos), grey peacock-pheasant (Polyplectron bicalcaratum), green peafowl (Pavo muticus), spot-billed pelican (Pelecanus philippensis), Oriental darter (Anhinga melanogaster), painted stork (Mycteria leucocephala), greater adjutant (Leptoptilos dubius), red-headed vulture (Sarcogyps calvus), mountain hawk-eagle (Nisaetus nipalensis), lesser fish eagle (Ichthyophaga humilis) and all six species of hornbill (Bucerotidae) living in mainland Southeast Asia.

"[11] In early 2018, Premchai Karnasuta, the president of the Italian-Thai Development PLC (ITD), one of Thailand's largest construction companies, was arrested in the sanctuary in possession of skinned carcasses of protected wild animals, including a black leopard,[12][13] a Kalij pheasant, and a common muntjac (also known as a barking deer), as well as three rifles and ammunition.

For at least 700 years, the Dawna-Tenasserim region has been home to Mon and Karen people, but burial grounds in Thung Yai and Huai Kha Khaeng Wildlife Sanctuary have not been systematically researched.

[19][18] Historical sources as well as local oral tradition suggest that settlement of Karen people in Thung Yai did not occur before the second half of the 18th century.

At that time, due to political and religious persecution in Burma, predominantly Pwo-Karen from the hinterlands of Moulmein and Tavoy migrated into the area northeast of the Three Pagodas Pass, where they received formal settlement rights from the Siamese Governor of Kanchanaburi.

During the first half of the 20th century, external political influences were minimal in Thung Yai and the Karen communities were highly autonomous regarding their internal affairs.

Due to strong logging and mining interests in the area, it was not before 1972 that the Huai Kha Khaeng Wildlife Sanctuary could be established, and Thung Yai resistance was even stronger.

After the military had taken power once again in October 1976, many of the activists of the democracy movement fled into peripheral regions of the country and some of them found refuge among the Karen people living in Thung Yai.

Pointing to the high value of Thung Yai for nature conservation and biodiversity, dam opponents on the national and international level raised the possibility of declaring the area a world heritage site.

This prestigious option would have been lost with a huge dam and reservoir in the middle of the two wildlife sanctuaries most promising to meet the requirements for a global heritage.

In the nomination, the "outstanding universal value" of the two sanctuaries is, in first place, justified with their extraordinary high biodiversity due to their unique position at the junction of four biogeographic zones, as well as with its size and "the undisturbed nature of its habitats".

[9] Even though the UNESCO nomination explicitly emphasizes the "undisturbed nature" of the area,[7] and notwithstanding scientific studies supporting traditional settlement and use rights of the Karen people in Thung Yai as well as the sustainability of their traditional land use system and their strong intention to remain in their homeland and to protect it,[20][24][25][26][27][28][29][18][30] governmental authorities regard the people living in Thung Yai as a threat to the sanctuary and pursue their resettlement.

During the 1980s and early-1990s, villages of the Hmong ethnic minority group were removed from the Huai Kha Khaeng and Thung Yai Naresuan Wildlife Sanctuaries.

The resettlement of the remaining Karen in Thung Yai was announced in the management plan for the sanctuary, drafted in the late-1980s, as well as in the proposal for the world heritage site.