The chemical composition of the essential oil produced by steam distillation of the inflorescences of naturally growing Anacamptis coriophora (L.) R. M. Bateman, Pridgeon & M. W. Chase subsp. fragrans (Pollini) R. M. Bateman, Pridgeon & M. W. Chase (Orchidaceae) from Kroumiria, north-west Tunisia was studied by GC-MS, which led to the identification of 19 volatile components, representing 97% of the oil. The main constituents were methyl-( E)-p-methoxycinnamate (29.3%), 13-heptadecyn-l-ol (18.6%), 2,5-dimethoxybenzyl alcohol (14.1%) and 4-(1,1,3,3-tetramethylbutyl)-phenol (9.0%). DPPH radical scavenging revealed a weak antioxidant activity. In addition, the antiproliferative effects were evaluated on BxPC3 human pancreatic carcinoma cells and on 2008 human ovarian cancer cells showing significant effect. This is the first report of the chemical composition of essential oils obtained from A coriophora subsp. fragrans inflorescences for North Africa. Further studies are needed to understand fully the possible mechanism of action behind the cytotoxic activity of the essential oil.