Bovine tick has caused losses in livestock production profitability in Brazil. However, tick control has caused resistance of these ectoparasites against utilized acaricides. Alternative tick controls have been utilizing plants as sources of effective botanical acaricides. Brunfelsia uniflora is a Brazilian plant with antimicrobial and antioxidant activity; however, there are no reports on its acaricidal activity. Therefore, this study aimed to evaluate the chemical composition of B. uniflora leaf ethanolic extract and its efficiency to control bovine tick in vitro and free-living stage ex situ. The crude leaf extract was analyzed by gas chromatographer coupled to mass spectrometer (GC-MS) with identification of 17 compounds. The major compounds were phytol (22.96%), 9,12,15-octadecatrienoic acid, ethyl ester (Z,Z,Z) (21.18%), hexadecanoic acid, ethyl ester (12.74%) and vitamin E (8.77%). The crude extract presented acaricidal activity in vitro against ingurgitated adult females, larvae and eggs of bovine tick. The LC99.9 for larvae was 103.21 mg mL-1 in in vitro tests and was 100% efficient for ex situ larva test (free-living stage). B. uniflora leaf extract is an alternative for the control of the bovine tick cycle, mainly in the free-living stage (non-parasitic stage) under field conditions.