Multiple drug resistance (MDR) in tumors, observed both in vivo and in vitro, is generally attributed to efflux-mediated reduced drug accumulation via mdr1/P-gp. Notoriously chemoresistant colorectal cancers exhibit the highest P-gp expression, account for 9% of all cancer deaths, and are the third most common cancer in men and women. P-gp, a 170kDa protein, contains twelve transmembrane segments with two ATP-binding domains used to extrude intracellular compounds of broad specificity against a concentration gradient. Studies concerning natural health products are becoming more prevalent in the search for safe anti-P-gp remedies and Hydrastis canadensis (goldenseal) was studied for its anti-cancer properties. Three forms of goldenseal from Sleepy Hollow Herb Farm (Dalton, Georgia) were tested in the Ames mutagenicity assay. The liquid extract, powder, and solid were tested in five ratios of root/rhizome:leaf/stem at five concentrations based on the minimum inhibitory concentrations to the tester Salmonella strain and compared to equivalent berberine amounts. All forms, ratios, and concentrations of goldenseal and berberine equivalents were found to be non-mutagenic. Goldenseal liquid in equal parts of root:leaf (GSL2:2) exerted the greatest activity with lowest toxicity and was assayed for its in vitro effects on colon cell viability, mdr1 gene expression, P-gp function, and ability to potentiate a chemotherapeutic drug. At 400μg/mL GSL2:2, HT-29 and doxorubicin-treated HT-29 (HT-29/Dox) but not CCD-18Co were significantly inhibited after 72 hours. Increasing amounts of GSL2:2 caused over 90% of cell death in CCD-18Co and HT-29; HT-29/Dox were still 50% viable even when treated with 6400μg/mL GSL2:2 after 72h. GSL2:2 at 400μg/mL led to significant increases in mdr1 gene expression in CCD-18Co after 24, 48, and 72 hours but did not significantly alter levels in HT-29 or HT-29/Dox. GSL2:2 at 100μg/mL and 400μg/mL reduced P-gp function in HT-29 and in HT-29/Dox at 400μg/mL. GSL2:2 potentiated the action of vinblastine in HT-29 and HT-29/Dox, though not in CCD-18Co. GSL2:2 was found to be non-mutagenic, possess anti-tumor properties, decrease P-gp function in colon cancer cells and potentiate a drug. Therefore it is plausible that GSL2:2 could be co-administered with a chemotherapeutic to cancer patients to overcome MDR and achieve treatment success.