The impact of cancer on lifespan is significantly increasing worldwide. Enhanced activity of drug efflux pumps and the incidences of the tumor microenvironment such as the apparition of a hypoxic gradient inside of the bulk tumor, are the major causes of chemotherapy failure. For instance, expression of Hypoxia-inducible factor (HIF-1α) has been associated with metastasis, resistance to chemotherapy and reduced survival rate. One of the current challenges to fight against cancer is therefore to find new molecules with therapeutic potential that could overcome this chemoresistance. In the present study, we focused on the bioactive plant flavonoid quercetin, which has strong antioxidant and anti-proliferative properties. We examined the efficacy of combined treatments of quercetin and the anti-cancer drugs gemcitabine and doxorubicin, known to specifically act on human pancreatic and hepatic cancer cells, respectively. Moreover, our study aimed to investigate more in-depth the implication of the multidrug transporter MDR1 and HIF-1α n chemoresistance and if quercetin could act on the activity of the drug efflux pumps and the hypoxia-associated effects. We observed that the anti-cancer drugs, were more effective when administered in combination with quercetin, as shown by an increased percentage of dead cells up to 60% in both 2D and 3D cultures. In addition, our results indicated that the combination of anti-cancer drugs and quercetin down-regulated the expression of HIF-1α and increased the expression levels of the regulator of apoptosis p53. Moreover, we observed that quercetin could inhibit the efflux activity of MDR1. Finally, our in vitro study suggests that the efficiency of the chemotherapeutic activity of known anti-cancer drugs might be significantly increased upon combination with quercetin. This flavonoid may therefore be a promising pharmacological agent for novel combination therapy since it potentializes the cytotoxic activity of gemcitabine and doxorubicin on by targeting the chemoresistance developed by the pancreatic and liver cancer cells respectively.