Abstract In search of a plant that exhibits osteogenic activity, Mimosa tenuiflora (M. tenuiflora) cortex represents the opportunity to create a biomaterial that, together with the chitosan, is osteoconductive and promote better and rapid regeneration of bone tissue. Thus, the composite of chitosan/M. tenuiflora cortex fabricated will have properties of biocompatibility and allow the osteoblast proliferation. Composites were developed with different concentrations of chitosan/M. tenuiflora cortex (w/w) using thermally induced phase separation technique (TIPS). To analyze the effects of composite on osteoblasts, primary cultures, each sample was collected on days 1, 3 and 7 after seeding. The evaluation of composites consisted of viability and proliferation tests in which we observed the metabolic activity of the cells using MTT reagent and determined the DNA concentration by means of fluorescence. The expression of the marker alkaline phosphatase (ALP) using p-nitrophenyl phosphate was examined, allowing the observation to the activity of proliferation and differentiation of osteoblastic cells. Moreover, an analysis of biomineralization was performed using scanning electron microscopy (SEM), energy dispersive spectroscopy, infrared spectroscopy and X-ray diffraction. The results showed that 80/20 chitosan/M. tenuiflora cortex biocomposite has the best performance with osteoblasts compared to biomaterials 100/0 and 70/30 chitosan/M. tenuiflora composites. Finally, it was determined that the composite of chitosan/M. tenuiflora cortex presents no cytotoxicity and increases the capacity of the osteoblasts to proliferate and differentiate.