The river mouth is at Vallejo, where the intertidal zone of fresh and salt waters flow into the Carquinez Strait and the San Pablo Bay.

The source begins as seasonal Kimball Canyon Creek in Robert Louis Stevenson State Park at an elevation of 3,745 feet (1,141 m) which descends the southern slope of Mt.

In the valley, it flows southeast past Calistoga, St Helena, Rutherford, Oakville and through Napa, its head of navigation.

Downstream from Napa, it forms a tidal estuary, entering Mare Island Strait, a narrow channel on the north end of San Pablo Bay.

The Napa River basin is estimated to have historically supported a spawning run of 6,000–8,000 steelhead, and as many as 2,000–4,000 coho salmon (Oncorhynchus kisutch).

[9] Although diminished, the Napa River basin continues to support a fish community of greater diversity than even the Sacramento and San Joaquin River systems, including a nearly intact community of sixteen native fish species, including Steelhead, fall-run Chinook salmon, Pacific (Lampetra tridentata) and river lamprey (Lampetra ayresi), hardhead (Mylopharodon conocephalus), hitch (Lavinia exilicauda), tule perch (Hysterocarpus traski), and Sacramento splittail (Pogonichthys macrolepidotus).

[6] White sturgeon (Acipenser transmontanus) and many other native and non-native fishes currently utilize the Napa River watershed.

[19] As part of urban renewal in the 1970s, a concrete cover was removed from culverted sections of the Napa Creek, re-exposing the water to daylight.

There is some debate as to whether this constituted one of the country's earliest "daylighting" projects, since the construction was undertaken with little thought to the river's ecology or restoration of riparian habitat.

[18] These keystone species have been shown to increase fish, bird and mammal abundance and diversity in California rivers and streams.