Objective The essential oil from the leaves of Ocimum kilimandscharicum (EOOK), collected in Dourados-MS, was investigated for anticancer, anti-inflammatory and antioxidant activity and chemical composition. Materials and methods The essential oil was extracted by hydrodistillation, and the chemical composition was performed by gas chromatography-mass spectrometry. The essential oil was evaluated for free radical-scavenging activity using the DPPH assay and was tested in an anticancer assay against ten human cancer cell lines. The response parameter (GI50) was calculated for the cell lines tested. The anti-inflammatory activity was evaluated using carrageenan-induced pleurisy in mice. Results The chemical composition showed 45 components with a predominance of monoterpenes, such as camphor (51.81%), 1,8 cineole (20.13%) and limonene (11.23%). The EOOK exhibited potent free radical-scavenging activity by the DPPH assay with a GI50 of 8.31 μg/ml. The major constituents, pure camphor (IC50 = 12.56 μg/ml) and mixture of the limonene: 1, 8 cineole (IC50 = 23.25 μg/ml) displayed a potent activity. The oral administration of EOOK (at 30 and 100 mg kg-1), as well as the pure camphor or a mixture of 1,8 cineole with limonene, significantly inhibited the carrageenan (Cg) induced pleurisy, reducing the migration of total leukocytes in mice by 82 ± 4% (30 mg kg-1 of EOOK), 95 ± 4% (100 mg kg-1 of EOOK), 83 ± 9% (camphor) and 80 ± 5% (mixture of 1,8 cineole:limonene 1:1). In vitro cytotoxicity screening against a human ovarian cancer cell line displayed high selectivity and potent anticancer activity with GI50 = 31.90 mg ml-1. This work describes the anti-inflammatory, anticancer and antioxidant effects of EOOK for the first time. 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