Collombon and Mesa went a set down against three-seed Ortega and Gonzalez, who made the final in last week's La Rioja Open.
Thursday's action at the Universidad Católica featured plenty of upsets, but none bigger than at centre court as Ortega and Gonzalez have now fallen in the round of 16 twice in the first three tournaments.
Prior to their match, Paula Josemaria and Ariana Sanchez were in action against Carmen Goenaga and Bea Caldera while Gemma Triay and Alejandra Salazar faced Marta Caparros and Marta Barrera.
Watch all the centre court highlights below!
Matches in chronological order:
Paula Josemaria (ESP)/Ariana Sanchez (ESP) (2) vs Bea Caldera (ESP)/Carmen Goenaga (ESP)
Alejandra Salazar (ESP)/Gemma Triay (ESP) (1) vs Marta Caparros (ESP)/Marta Barrera (ESP)
Marta Ortega (ESP)/Bea Gonzalez (ESP) (3) vs Alix Collombon (FRA)/Carla Mesa (ESP)
