The aim of this study was to evaluate the influence of seasonality on the chemical composition of the essential oil from Hyptis dilatata leaves, to perform biological activity assays such as antimicrobial, inhibition of acetylcholinesterase enzyme and to evaluate the toxicity of the essential oil using Artemia salina as indicator on the test. Hyptis dilatata leaves were collected in rainy and dry seasons, in the morning, in the afternoon and in the evening. It was extracted by hydrodistillation using the extractor of Clevenger condenser double Spell model. The essential oil analysis resulted in 22 chemical components. The major constituents of the dry and rainy periods were a-pinene (18.7% in the night and 12.9% in the afternoon), 3-carene (26.5% and 19.9% in the night), fenchone (43% and 33.7% in the morning) and ß-caryophyllene 9.1% in the afternoon and 6.1% in the morning. The essential oil in vitro inhibited the acetylcholinesterase enzyme in 99.9% in the afternoon (rainy period) and 96.4% in the morning (dry period). Between the dry and rainy seasons, the lowest LC50 microbial activity in vitro was obtained for leaves collected during rainy season, in the morning period testedagainst the bacterium Staphylococcus aureus (LC50 78.1 mg mL-1). The cytotoxic activity of the essential oil ofdilatata on Artemia salina showed LC50 results below 100 µg mL-1 and in the afternoon during the rainy period and at night in the dry period the results are above this value. Therefore, the chemical characterization and biological activities tests of essential oils showed promising results in the search for new active substances and the development of bioproducts of vegetable origin.