Dried parts of 75 medicinal plant species collected from different regions in Iran were assayed by the Dish Pack Method for volatile allelopathic activity, using Lactuca sativa (lettuce) as the test plant. The highest (60%) inhibition was observed for saffron (stigma of Crocus sativus), followed by Dracocephalum kotschyi, Solanum nigrum and Artemisia aucheri. Safranal was identified as the main chemical by Headspace Gas Chromatography-Mass Spectrometry (HS- GC-MS) analyses of saffron. Moreover, the EC50 of safranal was evaluated as 1.2 μg/L (ppb). This is the first report on allelopathic activity of safranal as a bioactive compound identified from saffron.