"FHI 360 (a US non-profit organisation) condemns in the strongest possible terms the abduction of three staff members and two contractors in Ngala, Nigeria," Christy Delafield, an FHI spokesperson said in a statement.
Ngala is located in Borno State, near the border with Cameroon.
"We are extremely concerned for our colleagues and call for their unconditional, immediate and safe return," she added.
The nationality of the aid workers wasn't immediately clear
Delafield said the staff members were working to provide life-saving medical care in the region.
"We are not sure who is responsible," said another humanitarian official who asked to remain anonymous.
He said the abduction took place Tuesday evening.
Both Boko Haram, which kidnapped hundreds of schoolgirls in Chibok in 2014, and ISWAP, linked to the Islamic State group, operate in the region.
More than 40,000 people have been killed since the northeast insurgency began in 2009, according to the UN. Two million more have been displaced from their homes.
The violence has spread to parts of neighboring Niger, Chad and Cameroon, prompting a regional military coalition to fight the militants.
