Carlina acaulis (Compositae) is traditionally used for food and medicinal purposes in central and southern Europe. Its root essential oil (EO), mainly composed by carlina oxide, is included in the BELFRIT botanical list of food supplements. It is also recognized as a potent mosquito larvicide. It is matter of concern whether this EO could be endowed with intrinsic toxicity to limit its use on a food level. Focusing on the insecticidal activity of this EO, we investigated the acute toxicity and sublethal effects on Musca domestica. In topical assays, the EO was extremely effective (LD50 = 2.74 and 5.96 μg fly-1, on males and females, respectively). The exposure to a sublethal dose (LD30) led to significant reductions of female longevity (LT50 = 6.7-9.0 days vs. control LT50 = 12.9-13.7 days). Treated females laid 2.5 times fewer eggs over control ones. F1 vitality decreased: F1 larvae and pupae showed high mortality, 2-4-fold higher over the control. The EO also showed high cytotoxicity on normal human fibroblasts (NHF-A12, IC50 = 9.4-14.2 μg mL-1 after 6-48 h). Overall, our findings support the employ of this EO for developing botanical insecticides. At the same time, they encourage food safety authorities to perform a full toxicological assessment for possible restrictions at food level.