Astragalus L. species are recognized for their prominent biological activities, making them useful in the treatment of several diseases. Astragalus spruneri (AS), a perennial prostrate herbaceous wild plant endemic to Southeast Europe and Turkey, remains insufficiently explored to date. The present study investigated total phenolic content, DPPH radical-scavenging activity, and in vitro cytotoxic activity of extracts from aerial parts of A. spruneri. We analyzed the potential of methanolic (ASM) and ethanolic (ASE) extracts to induce cell death in MCF-7 cells using the Incucyte® Cytotox NIR dye and bright-field microscopy. ASE of the leaf was richer in flavonoids, whereas ASM of the leaf displayed higher antioxidant activity than the stem. Live-cell analysis showed that ASE of the leaf exhibited the highest cytotoxic potency (EC50 = 125 μg/mL after 48 hours) and induced a time- and concentration-dependent increase in Cytotox NIR signal. Compared with the vehicle control, ASE of the leaf produced an earlier onset and more significant sustained cytotoxic activity in MCF-7 cells. Western blot analysis further demonstrated that AS extract modulated PUMA protein expression. The Cytotox NIR assay provided new insights into distinct extract- and tissue-dependent cytotoxic responses against MCF-7 cancer cells, with ethanolic leaf extracts exhibiting the most prominent effect. Thus, it can be concluded that A. spruneri serves as a valuable source of natural bioactive compounds with promising cytotoxic activity. Hence, further research is needed to confirm its anticancer potential and assess its safety profile.