He served as the first director of the New York Zoological Park, known today as the Bronx Zoo, and he was a pioneer in the early wildlife conservation movement in the United States.

After serving as a taxidermist at Henry Augustus Ward's Natural Science Establishment in Rochester, New York, he spent 1.5 years, 1877–1878 in India and Ceylon collecting specimens.

In a letter written to his superior at the Smithsonian, George Brown Goode, Hornaday reported that, "in the United States the extermination of all the large herds of buffalo is already an accomplished fact".

Among his several activities, he established one of the world's most extensive collections, insisted on unprecedented standards for exhibit labeling, promoted lecture series, and offered studio space to wildlife artists.

During Dr. Hornaday's tenure as director of the New York zoo, Ota Benga, a pygmy native of the Congo, was placed on display in the monkey house in September 1906.

Although, according to the New York Times, "few expressed audible objection to the sight of a human being in a cage with monkeys as companions", black clergymen in the city took great offense.

"[10] New York Mayor George B. McClellan, Jr. refused to meet with the clergymen, drawing the praise of Dr. Hornaday, who wrote to him: "When the history of the Zoological Park is written, this incident will form its most amusing passage.

[10] Hornaday decided to close the exhibit after just two days, and on Monday, September 8, Benga could be found walking the zoo grounds, often followed by a crowd "howling, jeering and yelling".

[10] Benga died by suicide in 1916 when his return trip to the Congo was delayed by World War I. Hornaday's became an advocate for preserving the American bison from extinction.

When the first large-game preserve in America was created in 1905—the Wichita National Forest and Game Preserve—Hornaday offered fifteen individuals from the Bronx Zoo herd for a reintroduction program.

As the historian Douglas Brinkley has described it, "What Upton Sinclair's The Jungle had been for meatpacking reform, Our Vanishing Wildlife was for championing disappearing creatures like prairie chickens, whooping cranes, and roseate spoonbills.

As he proclaimed with characteristic zeal in Our Vanishing Wildlife, "It is time for the people who don't shoot to call a halt on those who do; 'and if this be treason, then let my enemies make the most of it!

By 1915, the American Museum Journal declared that Hornaday "has no doubt inaugurated and carried to success more movements for the protection of wild animal life than has any other man in America.