Thirty-five different yacon (Smallanthus sonchifolius Poepp. & Endl) accessions were evaluated as potential alternative sources of fructooligosaccharides (FOS) and phenolic type natural antioxidants. FOS, total phenolics (TPC) and antioxidant capacity (AC) contents in the ranges of 6.4-65 g/100 g of dry mater (DM), 7.9-30.8 mg chlorogenic acid (CAE)/g of DM and 23-136 μmol trolox equivalente (TE)/g DM were found. Accession AJC 5189 sparked attention for its high FOS content while DPA 07011 for its high TPC and AC. In addition, the prebiotic effect of yacon FOS was tested in vivo with a guinea pig model. A diet rich in yacon FOS promoted the growth of bifidobacteria and lactobacilli, resulting in high levels of short chain fatty acids (SCFAs) in the cecal material and enhancement of cell density and crypt formation in caecum tissue, being indicative of colon health benefits. This study allowed identification of yacon cultivars rich in FOS, AC and/or FOS and AC for nutraceutical applications.