Abstract Extracellular vesicles (EVs) are nano-sized particles that are naturally released by cells. Recently, increasing attention has been directed toward EVs derived from natural sources such as plants and fruits. This is due to their sustainability and cost-effectiveness compared to mammalian-derived EVs. Microalgae are photosynthetic unicellular organisms that have also been reported to secrete EVs. In this study, we aimed to isolate EVs from the well-known microalga Chlorella vulgaris and evaluate their cytotoxicity in Jurkat T cells. EVs were isolated using a combination of ultracentrifugation and size-exclusion chromatography (SEC). Characterization was conducted using MicroBCA protein assay, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). Subsequently, the EV-enriched population was co-cultured with Jurkat T cells, and cell viability and apoptosis were assessed. Our results demonstrated that the EV-enriched population had a mean size below 200 nm. Moreover, the EVs did not cause any significant reduction in cell viability or induce apoptosis. Overall, our preliminary study has shown that microalgae-derived EVs are non-cytotoxic and are biocompatible in a mammalian system.