Abstract The present work is focusing on the technical viability of the reduced pressure steam distillation to perform the deterpenation of the Origanum majorana L. essential oil (EO), and the evaluation of antioxidant and antifungal activities. The EO and the obtained fractions (F1, F2 and F3) were characterized by GC/MS and FT-IR. The result showed that, the chemotype of the EO was terpin-4-ol (27.32%), γ-terpinene (15.67%) and α-terpinene (11.08%). However, during the deterpenation process the chemotype changed for F1 to γ-terpinene (27.53%), α-terpinene (21.15%) and sabinene (11.17%), for F2 to terpin-4-ol (54.39%) and cis -sabinen hydrate (10%) and for F3 to terpin-4-ol (48.6%) and β-fenchyl alcohol (23.84%). Moreover, the free radical scavenging method showing that the EO and its fractions did not present a strong antioxidant capacity. Regarding the antifungal activity, an experimental design with different methods was applied. Particularly, the deterpenated fractions showed the highest activity against Aspergillus niger with 100% and 85.09% of inhibition using the F2 and F3 respectively. However, the raw EO and the F1 presented the lowest antifungal activity with 36.56% and 8.76% of inhibition, respectively. In conclusion, this work demonstrates that reduced pressure steam distillation was an effective method to perform the fractionation of the raw EO from Origanum majorana L. Particularly, deterpenated fractions possess an interesting antifungal activity highlighting their possible uses in biotechnological fields and/or pharmaceutical industry.