Rhaponticoides iconiensis (Hub.-Mor.) M.V.Agab. & Greuter is one of the species of Asteraceae family. In this study, the α-amylase and α-glucosidase inhibition activity, the antioxidant, antimicrobial properties and the phytochemical composition of extract and sub-extracts from the leafy stem of R. iconiensis were evaluated. The antimicrobial effect of extracts was tested with the microdilution method and the antioxidant activity was investigated with DPPH·, ABTS·+ and FRAP tests. The phytochemical composition of active sub-extract carried out by LC–MS/MS. As a result, the highest α-glucosidase and α-amylase activity were found in ethyl acetate sub-extract (TSE) of R. iconiensis leafy stem extract (IC50 = 12.108 ± 0.582 µg/mL and 287.733 ± 0.325 µg/mL, respectively). Moreover, the highest total phenolic (312.001 ± 2.008 mgGAE/gextract) and total flavonoid content (242.007 ± 2.646 mgCA/gextract) was found in TSE extract too and this extract was found more active on DPPH· (IC50 = 0.249 ± 0.002 mg/mL), ABTS+· (at a concentration of 0.5 mg/mL equivalent to 0.542 ± 0.002 µM Trolox) and FRAP (at a concentration of 1 mg/mL equivalent to 3642.005 ± 2.646 mmol Fe2+) tests. Besides that, the flavonoids such as apigenin, naringin and patuletin derivatives were determined as the main compounds of active TSE sub-extract. This is the first report that these biological activities investigated and phytochemical compounds were identified in leafy stem extracts of R. iconiensis and it is thought that these studies will be shed light on future studies on this species.