Nigeria lost 1-0 to Ecuador in a friendly match played in the early hours of Friday.
It was the second consecutive loss under new head coach, Jose Peseiro.
An early goal by Villarreal left-back Pervis Estupinan saw the South Americans claim victory.
This comes after a 2-1 defeat to Mexico earlier in the week in another friendly game played in the US.
The three-time AFCON champions have now gone five straight games without a win, losing three and drawing two.
Up next for Peseiro and his team is the 2023 AFCON qualifiers against Sierra Leone and Sao Tome and Principe.