The European Centre for Constitutional and Human Rights (ECCHR), which has been supporting her, said Friday that the Swiss Federal Supreme Court had rejected the case .
Gladys Romero had been appealing an earlier court decision which ruled out an inquiry into allegations that Nestle could be responsible for the death of Luciano Romero, who was killed by Colombian paramilitaries in 2005.
The ECCHR said in a statement that the court had agreed with the previous ruling that the statute of limitations had been exceeded in the case.
Leonardo Jaimes, the lawyer representing Romero's widow, said: "We had hoped for more justice in Switzerland and a sign which compels the Colombian criminal justice system to investigate the Nestle managers in Colombia."
Nestlé had been accused of failing to take the necessary measures to protect Romero, a former employee of one of their subsidiaries, Cicolac.
He had worked for the company for many years when he was accused of collaborating with guerilla fighters, and received a number of death threats before being killed in Valledupar in the north of the country.
A number of paramilitaries were jailed for the assassination, but one former commander testified that the group had been financed by several companies, including Cicolac.
Nestlé has always denied the allegations.
When the case was first lodged in 2012 a spokesperson said that the firm had "never been associated with criminals" and was "in no way responsible, directly or indirectly, for the death of Luciano Romero."
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The company previously pointed out that Romero had stopped working for Cicolac three years before he was killed, and that he was murdered at a time when violence was rife across the country.
"Union leaders had been threatened, kidnapped and assassinated, as had members of our local team, other employees and members of their family," the company said.
The ECCHR said that together with the Colombian trade union Sinaltrainal and Romero's lawyers it is considering whether to take further legal action, including taking the case to the European Court of Human Rights.