Macarao is a major part of the ecological corridor that runs through the central section of the Cordillera de la Costa Mountain Range. The other protected areas that comprise this corridor are Avila National Park to the east of Macarao, and Pico Codazzi Natural Monument, Henri Pittier National Park, and San Esteban National Park to the west.

The faunal diversity of the Cordillera de la Costa Mountain Range is well represented in the park. The park is home to numerous mammal species including the red brocket deer (Mazama americana), the peccary (Tayassu sp.), the crab-eating fox (Cerdocyon thous), the red howler monkey (Alouatta seniculus) and the brown-throated sloth (Bradypus variegatus). Large cats like the jaguar (Panthera onca), the puma (Puma concolor) and the ocelot (Leopardus pardalis) are present as well. There are also reports from locals of the presence of bush dogs (Speothos venaticus).

Macarao is home to at least 263 bird species, 12 of which are endemic to Venezuela. They include the tapaculo (Scytalopus caracae), the handsome fruit eater (Pipreola formosa), the rufous-tailed ant thrush (Chamaeza ruficauda), the grooved-billed toucanet (Aulacorhynchus sulcatus) and the violet-chested hummingbird (Sternoclyta cyanopectus). Globally threatened species such as the helmeted curassow (Pauxi pauxi), the Venezuelan bristle-tyrant (Phyloscartes venezuelanus), and the Venezuelan wood-quail (Odontophorus columbianus) are also present.

The flora is highly diverse. In the forests of Macarao, the genus Guarea, Gustavia, Inga, Ocotea and Tabebuia are well represented. The most common tree species are Cedrella americana, Pithecelobium saman, Erithrina poeppigiana, Tabebuia chrisantha, Cordia alliodora, and the fern Equisetum giganteum. The palms Wettinia praemorsa and the endemic Ceroxylom interruptum are common, as are the epiphytes of the genus Tillandsia and Epidendrum.