Todd Davis, 44, and Joshua Keathley, 36, both of Lovelock, are alleged to have shot the horses on or about November 28, 2009, in Washoe county, Nevada.
Representatives of the Cloud Foundation, a wild horse advocate group, were in court for the pair's appearance.
Davis and Keathley face up to a year in jail and a fine of up to $US100,000 if found guilty.
Federal authorities allege that the pair maliciously caused the death and harassment of five wild free-roaming horses by shooting them.
The trail has been set for June 22.
The Bureau of Land Management, which is responsible for the management of wild horses in the US, had offered a $US10,000 reward for information in the case, and the Humane Society had added $US2500.
Wild horse advocates have expressed their concern that each defendant has been charged with only one count of causing the death of five wild horses. They believe each should face five counts.