The Niagara Falls Museum had humble beginnings. In 1827, the first museum contained Thomas Barnett's own cabinet of curiosities. Although the details were not documented, the collection was likely composed of a number of mounted animals of local origin, combined with a smattering of Native American artifacts. Barnett's collection rapidly grew.

Prior to 1844, an account of the museum's contents stated that there were over 5,000 items, including bipeds, quadrupeds, birds, fish, insects, reptiles, shells, minerals, and Native American curiosities. Through the first fifty years of its existence, the Niagara Falls Museum continued to acquire similar artifacts through the diligent efforts of the Barnett family and their associates.

In 1854, Sydney Barnett (son of Thomas Barnett) made the first of his three trips to Egypt (two by himself and one with Dr. J. Douglass of Montreal) and purchased four mummies and a host of other Egyptian antiquities. In 1857, mastodon remains were discovered in the St. Thomas, Ontario and later placed in the museum. In 1859 an inventory of the museum's contents included, in addition to the previously mentioned artifacts, an egg collection, ancient and modern coins, Japanese and Chinese relics.

In 1873, the Barnetts purchased the remains of a large whale, the 40-ft magnificent humpback whale skeleton. Barnett and his son Sydney, who assisted with the Museum, were both accomplished taxidermists, preparing specimens for the museum as well as trade and sale to other institutions. Sydney Barnett, as well as being a Colonel in the army, was a poet, writer, and inventor, and was referred to at the time as "Gadget". Also, with the growing interest in the 'Wild West', Sydney Barnett began organizing a Wild West Show and Grand Buffalo Hun in 1872t. He originally contacted "Buffalo Bill" Cody to feature lasso men and over 100 Pottawatomie Indians in a large buffalo hunt. Problems arose when the US Government would not allow the Indians off the reservation. This show was then changed to feature General Custer's scout "Wild Bill" Hickock as master of ceremonies, assisted by local Woodland Indians of the Tuscarora and Cayuga Nations.

In 1888, no suitable location could be found in Canada so it was relocated to Niagara Falls, New York. The Davis family established an Art Gallery in the museum in 1891. During the early years of their proprietorship, five more Egyptian mummies were purchased, along with the entire collection from the celebrated Wood's Museum of Chicago. While acquiring a few new exhibits, the museum also lost or disposed of others. There was also exchange of artifacts and specimens between the Niagara Falls Museum and P.T. Barnum and these early North American institutions have been documented.

In 1892, the museum's living display came to an end because of complaints from area residents about the noises and the odors. A number of artifacts displayed in Buffalo at the Pan American Exposition of 1901 were acquired by the Niagara Falls Museum. A giant Sequoia tree that was reportedly felled on the Eel River, Humboldt County, California, on February 14, 1893, was a highlight in the Forestry Building at the Exposition. Its circumference was seventy-seven feet, making it one of the largest trees ever cut down in the world. Also given to the museum was a shell and coral collection gathered by Dr. L. Agassiz, of Harvard University. The Exposition contained a wealth of artifacts, and although only the above artifacts have been documented, there may be items in the museum's Eskimo, Oriental and South Sea Island display originating from the Exposition.

Thomas Barnett, died in 1890 in Niagara Falls, Canada, founder of Canada's oldest museum, is considered Canada's first "Museum Man". Both Thomas and Sydney passed from this world without ownership of the museum they founded and built. The museum collection was owned by the Sherman family until May 1999. The entire collection was purchased by a private collector in Toronto, whose hopes are to one day resurrect the dream that Thomas Barnett started.