Mimosa tenuiflora (Willd.) Poir., known as "Jurema-preta", is a species of ethnobotanical relevance, used in traditional medicine. The northeastern backland is characterized by climatic variability, which makes it essential to carry out studies that assess the seasonal influence on the chemical profile of native species. The aim of this study was to evaluate the seasonal variation in the chemical constituents of M. tenuiflora over a 24-month period. Plant collections were made bimonthly in the municipality of Petrolina, Pernambuco, Brazil. The plant material was dried, pulverized, and extracted with ethanol (1:10, w/v) for 72 h under magnetic stirring. The analyses included the quantification of secondary metabolites, the development of a method using high-performance liquid chromatography with a diode array detector for the quantification of N,N-dimethyltryptamine (DMT), and multivariate statistical analysis using principal component analysis. The results indicated that phenolic compounds, especially flavonoids, are associated with water stress, radiation, and temperature. DMT production showed a negative correlation with radiation and temperature, but not with precipitation. Total alkaloids and yield showed little correlation with climatic variables. This study reinforces the importance of seasonality in the planning of plant collection and the pharmacological use of M. tenuiflora.M. tenuiflora, "Jurema preta", seasonality, HPLC-DAD, PCA