The tick Rhipicephalus microplus affects cattle health, with production loss in tropical and subtropical regions. Moreover, the use of commercial acaricides has been reduced due to the resistance of this parasite. Although alternatives such as plant bioactive molecules have been sought, essential oils present variations in their chemical constituents due to environmental factors, which can interfere with their acaricidal activity. The objective of the present study was to evaluate the seasonal influence of the essential oil of Ocimum gratissimum and its major constituents on acaricidal activity against R. microplus larvae. A high-yield essential oil of O. gratissimum and its major constituents were used, and a plant with a thymol-type oil was selected for seasonal analysis and acaricidal activity against R. microplus. Gas chromatography (GC) and GC-mass spectrometry (MS) were employed to identify 31 oil constituents (average yield of 6.26%). The main compounds were found to be thymol (33.4 to 47.9%), γ-terpinene (26.2 to 36.8%), and p-cymene (4.3 to 17.0%). Concerning acaricidal activity, the December (LC 0.84 mg/mL) and September (LC 1.58 mg/mL) oils obtained in the dry season were the most active, and assays performed with commercial standards revealed LC values of p-cymene, thymol, and γ-terpinene of 1.41, 1.81, and 3.08 mg/mL, respectively. Overall, lower acaricidal activities were found for oils produced from plants harvested in the rainy season. The results showed that seasonal variation in the chemical composition of the O. gratissimum essential oil influences its acaricidal activity. The seasonal variations in the thymol-type essential oil of O. gratissimum can represent an important strategy for the control of R. microplus.