Portulaca johnstonii is a rare purslane endemic to the arid zones of northern Mexico. Plants grow in calcareous and chalky soils with low humidity. This study aimed to characterize the phytochemical composition and evaluate the antioxidant activity of P. johnstonii Henrickson (Portulacaceae). Species identification was confirmed based on morphological and molecular characteristics. Antioxidant capacity was detected in the water and ethanolic extracts, and the water extract showed a higher antioxidant capacity. Metabolites were identified using mass spectrometry, and 93 phytochemical compounds were tentatively identified in the aqueous and ethanolic extracts. Terpenes, alkaloids, phenolics, glycosides, and their derivatives were also identified. The phytochemical composition of this species is similar to that of other species of Portulaca. This report on P. johnstonii phytochemical characterization and antioxidant activity provides information for evolutionary and ecophysiological studies of the genus and conservation strategies for the species and its environment. In addition, these findings support the potential of P. johnstonii as a promising model for studying antioxidant responses to environmental stressors and underscore its value as a potential source of bioactive compounds adapted to arid environments.