This work investigated the chemical composition of essential oils (EOs) obtained from the aerial parts of three Inula species of the Tunisian flora as well as their biological properties. Inula viscosa essential oil (IVEO) contained mainly α-costic acid (65.4%). The EOs of Inula graveolens (IGEO) and Inula crithmoides (ICEO) were characterized by high contents of p-cymene derivatives while ICEO stands out by high levels of modheph-2-ene (16.5%) and tiglic esters (14.1%). Moreover, a notable antioxidant power was proved by the ORAC test for the three EOs. Finally, the cytotoxic activity was evaluated on two healthy cell lines (CCD45-SK, HUVECs) and two cancer cell lines (HepG-2, MCF-7) and it appeared that the EOs were particularly effective on MCF-7 human breast cancer cells with LC50 equal to 79, 16, and 63 µg. mL−1 for IVEO, IGEO, and ICEO, respectively.