Approximately 400 km of the river is within the reserve, forming the core area. Apart from the Ganges River Dolphin (Platanista gangetica), the other inhabitants of the sanctuary include magar (crocodile) and gharial (Gavialis gangeticus).
The Critically endangered Gharial crocodile and the Red-crowned roof turtle live here, and together with the endangered Ganges River Dolphin are the keystone species of the sanctuary. Other large threatened inhabitants of the sanctuary include Muggar crocodile, Smooth-coated Otter, Striped Hyaena and Indian Wolf.
Chambal supports 8 of the 26 rare turtle species found in India, including Indian narrow-headed softshell turtle, Three-striped roof turtle and Crowned river turtle. Other reptiles who live here are: Indian flapshell turtle, Soft Shell turtle, Indian roofed turtle, Indian tent turtle and Monitor lizard.
Mammals of less concern who live here include: Rhesus Macaque, Hanuman Langur, Golden Jackal, Bengal Fox, Common Palm Civet, Indian Small Mongoose, Indian Grey Mongoose, Jungle cat, Wild Boar, Sambar, Nilgai, Blackbuck, Indian Gazelle (Chinkara), Northern Palm Squirrel, Porcupine, Indian Hare, Indian Flying Fox and Hedgehog.
The National Chambal Sanctuary is listed as an important bird area (IBA) IN122 and is a proposed Ramsar site. At least 320 species of resident and migratory birds inhabit the sanctuary. Migratory birds from Siberia form part of its rich avian fauna.
Vulnerable bird species here include the Indian skimmer, Sarus Crane, Pallas's Fish Eagle and Indian Courser. The Pallid Harrier and Lesser Flamingos here are near threatened. Winter visitors include Black-bellied Terns, Red-crested Pochard and Ferruginous Pochard and Bar-headed Goose. Other species include Great Thick-knee, Greater Flamingos, Darters, and Brown Hawk Owl.
Common plants in the sanctuary include Khair (Mimosa catechu Acacia catechu), Palash (Flame of the Forest Butea monosperma), Churel (Indian Elm Tree Holoptelia integrifolia), Ber (Indian Plum Ziziphus mauritiana) and Grassy patches on both sides of the river.