The Chitwan valley features sub-tropical forest. Roughly 70% of the park is covered by sal forest (shorca rebusta), a moist deciduous vegetation typical to terai region. The remaining types include grassland (20%), riverine forest (7%), and sal with chirpines (30%) forest which is found on the churey range towards south border of the park.

The grassland is formed of a diverse and complex community with over 50 plant species. The saccharum species, often called Elephant grass, can reach 8m. in height. The shorter grasses such as imperata are useful for roof thatching.

There are more than 43 mammal species in Chitwan National Park. The park is especially famous for protection of the endangered one-horned rhinoceros, whose estimated population is 460. The park also secures populations of other endangered species such as tiger Gaurs (Largest of all bovines), wild elephants, four-horned antelopes, striped hyenas, pangolins, gangetic dolphins, gharial crocodiles, monitor lizards and pythons. Other animals found in the park include four different types of deer: Sambar, Chital, hog deer and barking deer. Sloth bears, common leopards, ratels, palsm civets, tiger civets (Linsang), wild dogs, langurs and rhesus monkeys also inhabit the park.

There are over 450 species of birds in the park, among which the endangered birds are the Bengal floucan, giant hornbell, lesser floucan, black stork and white stork. A few of the commonly seen birds are peafowl, red jungle fowl, parakeets and different species of egrets, herons, kingfisher, flycatchers and woodpeckers. The best time for bird watching is March and December.

In addition, over 45 different species of reptiles and amphibian including marsh muggers, crocodiles, cobras, green pit vipers and various species of frogs and tortoises can be found in the park.

Chitwan National Park is also actively engaged in the scientific study of several species of wild flora and fauna.