Animals' Angels and Animal Law Coalition made the request of the Government Accountability Office (GAO), alleging violations were found to have occurred.
The group argues there were violations of US Department of Agriculture (USDA) regulations over humane treatment.
The request for a GAO investigation comes in the wake of a Freedom of Information Act request for documents which were subsequently released by the USDA.
The coalition says several hundred photos taken during 2005 at US slaughterhouses by USDA investigators indicated alleged breaches.
Though horse slaughter ended in the US in 2007, American horses are still sold for slaughter in Mexico and Canada.
Proponents of horse slaughter argue for the re-opening of horse slaughter plants, saying US laws protect horses from cruelty.
However, the coalition argues the documents and photos released by the USDA challenge such claims.
Photographs document horses with legs ripped off, eyeballs hanging on their cheeks and other severe injuries.
Earlier GAO documents raise additional disturbing humane issues. A 2004 GAO report found horses were slaughtered while conscious, which are considered significant violations of the Humane Methods of Slaughter Act.
Sonja Meadows, Executive Director of Animals' Angels, a Maryland-based animal welfare organisation, said: "We are asking the GAO to thoroughly examine the ineffectiveness of laws in preventing documented cruelty and inhumane practices."
The evidence suggested horse slaughter was not, "as horse slaughter advocates would have the public believe, a service motivated by altruistic concerns or humane practices".
"We believe the GAO's report will be the definitive wakeup call to the new USDA Secretary Tom Vilsack and the Obama administration to push for an immediate ban on horse slaughter."
Horses are slaughtered for consumption overseas, primarily in parts of Europe and Asia. A USDA study shows that 92 per cent of American horses at slaughter are young, healthy and without behavioral problems, challenging claims that horse slaughter provides a service in disposing of horses that are old, lame, sick or suffering from behavourial problems.