The essential oil extracted from the fruits of Cicuta virosa L. var. latisecta Celak was tested in vitro and in vivo against four foodborne fungi, Aspergillus flavus, Aspergillus oryzae, Aspergillus niger, and Alternaria alternata. Forty-five different components accounting for 98.4% of the total oil composition were identified by gas chromatography-mass spectrometry. The major components were γ-terpinene (40.92%), p-cymene (27.93%), and cumin aldehyde (21.20%). Antifungal activity was tested by the poisoned food technique against the four fungi. Minimum inhibitory concentration against the fungi was 5 μL/mL and percentage inhibition of mycelial growth was determined at day 9. The essential oil had a strong inhibitory effect on spore production and germination in all tested fungi proportional to concentration. The oil exhibited noticeable inhibition on dry mycelium weight and synthesis of aflatoxin B₁ (AFB₁) by A. flavus, completely inhibiting AFB(1) production at 4 μL/mL. The effect of the essential oil on inhibition of decay development in cherry tomatoes was tested in vivo by exposing inoculated and control fruit to essential oil vapor at a concentration of 200 μL/mL. Results indicated that the essential oil from C. virosa var. latisecta (CVEO) has potential as a preservative to control food spoilage.