Anemia tomentosa var. anthriscifolia is a very aromatic fern with ornamental potential. The major constituent of the volatile fraction of this plant is the triquinane sesquiterpene (-)-9-epi-presilphiperfolan-1-ol, which displays in vitro antibacterial activity against Mycobacterium tuberculosis. In order to investigate the chemical composition of this volatile fraction in relation to their triquinane sesquiterpenes upon in vitro multiplication, the tissue culture of this plant was established. Gametophytes and early sporophytes were successfully developed from spores inoculated in MS medium without growth regulators. Volatiles of both in vitro and wild plants were extracted by simultaneous distillation and extraction (SDE) and analyzed by gas chromatography/flame ionization detector (GC/FID) and GCâmass spectrometry (GC/MS). Twenty-seven constituents were evidenced in the volatiles of the wild plant, accounting for 93.4% of its total composition, whereas only seven constituents accounted for 97.0% of the total composition of the volatiles of the in vitro plants, respectively. The volatiles from the in vitro plants were composed mainly of monoterpenes (79%), whereas those of the wild plants comprised mostly sesquiterpenes (97.5%). The major constituents from in vitro plants are Î±-pinene (20.7%), trans-pinocarveol (31.0%) and pinocarvone (27.3%), whereas the major constituents from wild plants are silphiperfol-6-ene (11.7%), 9-epi-presilphiperfolan-1-ol (31.3%) and presilphiperfolan-8-ol (21.2%).