We have previously shown that the two sesquiterpene lactones, salograviolide A (Sal A) and iso-seco-tanapartholide (TNP), isolated from the Middle Eastern indigenous plants Centaurea ainetensis and Achillea falcata, respectively, possess selective antitumor properties. Here, we aimed to assess the anticancer effects of the separate compounds and their combination, study their potential to generate reactive oxygen species (ROS), and investigate their underlying antitumor mechanisms in human colon cancer cell lines. Cells were treated with Sal A and TNP alone or in combination, and cell viability, cell cycle profile, apoptosis, ROS generation and changes in protein expression were monitored. Sal A and TNP in combination caused 80% decrease in HCT-116 and DLD-1 cell viability versus only 25% reduction when the drugs were used separately. The antitumor mechanism involved triggering ROS-dependent apoptosis as well as disruption of the mitochondrial membrane potential. Further studies showed that apoptosis by the Sal A and TNP combination was caspase-independent and that ERK, JNK and p38 of the serine/threonine MAPKs signaling pathway were involved in the cell death mechanism. Taken together, our data suggest that the combination of Sal A and TNP may be of therapeutic interest against colon cancer.