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An essential oil complex (EOP) of Cymbopogon martinii with β-cyclodextrin (β-CD) was evaluated as a strategy to stabilize the essential oil of Palmarosa and increase its bioactivity. The complexation methods used were co-precipitation and kneading, and, for comparison, physical mixing was carried out. To confirm the molecular inclusion, techniques were combined for the characterization of the inclusion complex, such as Fourier Transformer Raman Spectroscopy (FT-Raman), Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR), Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA), which suggested the formation of the EOP-β-CD complex with complexation efficiency through the co-precipitation and kneading methods of 77.68% and 47.74%, respectively. Stability of the free and complexed EOP in relation to major compounds (linalool, geraniol, geranyl acetate and caryophyllene) during 14 days of storage was analyzed through Headspace Gas Chromatography coupled with Mass Spectrometry. Geraniol showed stability over the period of evaluation and increased availability when complexed by the Co-precipitation and kneading complex. Furthermore, the antifungal and antitumor activity of the EOP-β-CD complex was evaluated, showing reduced mycelial growth against Aspergillus flavus and Fusarium verticillioides strains compared to the free EOP (percentage of inhibition to toxigenic fungi A. flavus EOP-KM: 29.41%; EOP-CP and EOP-PM: 24.37 and 14.28%, respectively, and, the free EOP with 11.76%). In addition, an interesting activity against tumor cells (HeLa and HT-29) was observed against the tested complexes, which emphasizes the importance of the biotechnological use of β-CD for the stability of natural compounds.