The next display room is the Native American and Pioneer Room. The room houses an extensive collection of arrowheads, spear points, and other artifacts, all arranged by prehistoric periods. Pioneer artifacts include a split rail fence and tools, including a grain cradle, broad axe and froe, drawing knife, maul and more.

There are also what's thought to be the remains of dugout canoe, a Kentucky "Hog" Rifle, daguerreotype photo from Civil war area, oxen shoes, book published in 1794 containing act that created the state of Kentucky in 1792, various photos and maps pertaining to the First House at Kentucky built by Dr. Thomas Walker, Daniel Boone's trek through Knox County, and more.

One corner of the room features items from the John Allen Owens House (1894-1977), which at one time housed a museum. Owens was the first depot agent and an avid archeologist with items from old Middleton's Station in Knox County that are now housed at Levi Jackson State Park.