Randia echinocarpa, an endemic plant to Northwest Mexico, is used as food and in traditional medicine, and several of its biological activities have been demonstrated (antioxidant, antimutagenic, antidiabetic, and immunomodulatory). Plant tissue culture is a safe and scalable system for plant propagation and production of bioactive compounds. Therefore, this study aims to establish protocols for seed germination and callus culture of R. echinocarpa and to evaluate the antioxidant activity of methanol extracts (ME) of plantlets and calli via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods. Seeds were cultured in media with different concentrations of Murashige and Skoog (MS) salts and sucrose, and a higher germination rate and plantlet growth was observed in half-strength MS medium with 15 g L⁻¹ of sucrose. Calli were obtained from cotyledon and hypocotyl explants cultured in MS media with different concentrations of benzyl aminopurine (BAP) and indole-3-acetic acid (IAA). All treatments induced callus formation in 100% of explants; however, the medium containing 1 mg L⁻¹ BAP + 1 mg L⁻¹ IAA was selected because it produced calli with higher biomass and friable texture. The ME of cotyledons showed the highest antioxidant activity values (μmol Trolox per 100 g dry weight) in DPPH (345.5) and ABTS (1166.4) assays, whereas the ME of calli from hypocotyls showed a higher antioxidant activity than the ME of calli from cotyledons in both antioxidant assays. The tissue culture protocols established here will be useful for R. echinocarpa germplasm conservation and propagation, as well as for the production of bioactive compounds.