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Poincianella pyramidalis is an endemic Brazilian species that grows in semi-arid region (Caatinga) and employed by the local population as folk medicine. This species is characterized by the presence of bioactive flavonoids and biflavonoids, among them amentoflavone and agathisflavone. This work describes the production of amentoflavone and agathisflavone in P. pyramidalis calli by modifying the culture medium by employment of biotic and abiotic elicitors. Foliar explants were inoculated in Murashige and Skoog (MS) medium with different carbon sources (sucrose, glucose and fructose), combined with different 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations and maintained in the presence and absence of non-continuous light. The bioflavonoids produced were quantificated by HPLC/DAD from the organic extracts. The obtained results showed that the elicitors under study alter the production of interest compounds and the callus biomass formation. The addition of 30 g L-1 of sucrose + 5 mg L-1 of 2,4-D provided a higher accumulation of amentoflavone (16.44 mg L-1) and agathisflavone (0.58 mg L-1). The absence of light is ideal for the growth of callus biomass and biflavonoid production.