Rosa rugosa is widely used as a health food and medicine due to its broad pharmacological properties. Although the bioactivities obtained from plant materials is related to the type and amount of phytochemicals in each extract, no systematic information is available on the organ-dependent bioactivities of R. rugosa. Here, the antioxidant, anticancer, and anti-inflammatory activities of R. rugosa stem, leaf, flower, and fruit ethanol extracts were evaluated. Overall, the stem extract exhibited the highest levels of DPPH radical scavenging activity, ferric reducing power, and oxygen radical antioxidant capacity compared with other organ extracts, whereas leaves contained potent anticancer compounds that were particularly effective against A549 cells. Additionally, the leaf extract inhibited the MEK/ERK signaling pathway, resulting in the transcriptional repression of pro-inflammatory mediators in LPS-stimulated RAW 264.7 cells. Furthermore, significant correlation between phytochemical content and bioactivities indicated that phenolic compounds play as a major antioxidant compound of R. rugosa. Taken together, these findings suggested that the spatial distribution of the phytochemicals contributed to the biological activities of R. rugosa. Given that R. rugosa fruits and flowers are already being used in health foods and medicine, these results indicate that the leaves and stems of R. rugosa should also be included and used as natural sources of antioxidant, anticancer, and anti-inflammatory agents.