Speaking alongside Mexican President Enrique Pena Nieto during a state visit to Mexico on Thursday, Erdogan said the silence of Obama, Vice President Joe Biden and Secretary of State John Kerry was "telling" and they should take a position following such acts.
"If you stay silent when faced with an incident like this, and don't make a statement, the world will stay silent towards you," Erdogan said, in the latest sign relations between him and the White House have become strained.
The three Muslims were shot dead on Tuesday near the University of North Carolina campus in an incident police said was possibly a hate crime, Al Jazeera reported.
The White House said on Wednesday it would await the results of the police investigation before commenting.
Newlywed Deah Barakat, 23, a University of North Carolina dental student, his wife Yusor Mohammad, 21, and her sister, Razan Mohammad Abu-Salha, 19, a student at North Carolina State University, were gunned down on Tuesday in a condominium about three kilometres from the UNC campus in Chapel Hill.
Police charged the couple's neighbour, Craig Stephen Hicks, 46, with murder.
Investigators said initial findings indicated a dispute over parking prompted the shooting but they were looking into whether Hicks was motivated by hatred towards the victims because they were Muslim.
Turkey, a European Union candidate nation and member of the NATO military alliance, is a key US ally in the fight against the Islamic State in Iraq and the Levant (ISIL).
But Erdogan has become increasingly outspoken about what he sees as rising Islamophobia in the West.
Last year, Erdogan said his relations with Obama had become strained and that he no longer spoke directly with him as he was disappointed by a lack of US action over the war in neighbouring Syria.
Erdogan said he instead spoke with Biden over issues such as Iraq.
Despite working together to combat ISIL, differences have arisen between the US and Turkey over how best to tackle the rebels
More than 400 people held a gathering in the US capital of Washington DC to pay tribute to three young Muslims who were murdered in North Carolina this week.