To determine the effect of season and sampling location on the acaricidal activity of Petiveria alliacea against acaricides resistant Rhipicephalus microplus tick, methanol extracts of leaves and stems collected during the dry and rainy season at three sites in Yucatan, Mexico were used. Adult specimens of R. microplus ticks were collected to rear larvae and perform larval bioassays. For testing the acaricidal activity of plant extracts, larval immersion technique using different concentrations, namely: 20, 10, 5 and 2.5% was used. Extracts collected during the dry season exhibited the following 50% lethal concentrations (LC50): 4.4% and 1.7% for leaves and stems (respectively) from site 1, 8.2% and 2.2% for samples from site 2, and 18.3% and 22.4% for site 3. Extracts from samples collected during the rainy season showed the following LC50 values: 7.2% and 6.6% for leaves and stems (respectively) from site 1, 15.8% and 7.5% for samples from site 2, and 10.6% and 38.7% for site 3. In addition, gas chromatography-mass spectrometry run for stem samples showed that benzyldisulfide (BDS) and benzyltrisulfide (BTS) were present at higher concentrations in stems of dry season from sites 1 and 2. We conclude that the acaricidal activity of leaf and stem extracts from P. alliacea on resistant larvae of R. microplus is contingent upon the season and site of sampling. Extracts collected on dry season showed the highest acaricidal activity against R. microplus larvae. Site 1 showed the highest acaricidal activity across both seasons. The stem of P. alliacea performs better to control R. microplus, and higher concentration of BDS was observed in stem extracts collected on dry season in most locations. Further studies are needed to address the influence of edaphic and climatic factors on the phytochemical composition of plants.