The present study was conducted to elucidate the composition, antifungal and anti-inflammatory activities and cytotoxicity of Daucus carota subsp. gummifer essential oil. Aerial parts of the plants were hydrodistilled and the oil analyzed by GC and GC-MS. The oil was characterized by high contents of monoterpenes (83.9%), the major compounds being geranyl acetate (37.0%) and α-pinene (30.9%). The daucane sesquiterpene, carotol, was also found in relatively high amounts (11.0%). For the antifungal activity, minimal inhibitory and minimal lethal concentrations (MIC and MLC, respectively) were determined against several pathogenic fungi strains. The oil was particularly active against dermatophytes and Cryptococcus neoformans, with MIC values ranging from 0.32 to 0.64 μL/mL. Concerning the anti-inflammatory potential, the oil demonstrated a strong anti-inflammatory activity by inhibiting nitric oxide (NO) production in both lipopolysaccharide (LPS)-triggered macrophages and microglia cells. NO scavenging activity was also assessed using the NO donor S-nitroso-N-acetyl-d,l-penicillamine and a significant effect was disclosed. To assure that the bioactivity of the oil was achieved without detrimental effects to cells, the toxicity of D. carota subsp. gummifer oil was investigated using the MTT assay in several mammalian cell lines: macrophages (Raw 264.7), keratinocytes (HaCat), hepatocytes (HepG2) and microglia (N9). Interestingly, we detected a cytotoxic effect only for the highest concentrations of the oil, thus assuring a safe toxicological profile at bioactive concentrations.