Brett Knight pleaded not guilty to second-degree murder, aggravated harassment and other charges in the unsealing of a six-count grand jury indictment.
The charges stem from the September 24 killing of Ross Reisner in East Setauket. Prosecutors say one of the bullets also grazed the forearm of Reisner's partner, Kevin Murray.
Knight, who was arrested October 10 in Tennessee by Suffolk County homicide detectives and U.S. marshals, had previously rented a basement apartment from the two men. Reisner and Murray filed a harassment complaint against Knight last month.
Prosecutors allege the suspect said he wanted to kill both men.
The 50-year-old Reisner was a fixture in Eastern Suffolk County's equestrian community.
Knight is being held without bail.
The shooting spurred a nationwide search for Knight, who had purchased a motorcycle and was said to be traveling throughout the country.