Natural products are historically regarded as the main sources of bioactive compounds to fight the most diverse diseases; among them, propolis deserves to be highlighted due to several biological activities, such as antioxidant, anti-inflammatory, healing, antibacterial and antileishmanial activity. In this study, the antioxidant and antipromastigote activities and the cytotoxicity of propolis of the Scaptotrigona aff. postica stingless bee were evaluated. Propolis extract was analyzed by infrared spectroscopy, thermogravimetry and spectrophotometry for total polyphenols and flavonoid contents. Antioxidant activity was assessed spectrophotometrically by the DPPH, ABTS and FRAP methods, while antipromastigote activity and cytotoxicity were assessed by the MTT assay. The infrared spectra showed the characteristic bands of both phenols and alcohols. The thermogravimetry study revealed two events and thermal stability around 370 °C, while differential scanning calorimetry showed two endothermic events and an exothermic event. Total polyphenol and flavonoid contents were 21.29 ± 0.003 mg/g and 8.17 ± 0.00 mg/g, respectively, and the antioxidant activities identified by the DPPH, ABTS and FRAP methods were 910.09 ± 0.0264 µM Trolox (IC50 18.9 ± 0.01 µg/mL), 859.97 ± 0.0854 µM Trolox (IC50 692 ± 0.01 µg/mL) and 1613.67 ± 0.2610 µM Trolox (IC50 80 ± 0.1 µg/mL), respectively. The satisfactory antioxidant activity of propolis can be related to the high content of phenolic compounds, which adds value to this product and can contribute to the development of meliponiculture in the Brazilian state of Pará. The propolis extract caused a toxic effect on promastigote forms of Leishmania amazonensis at all concentrations tested, with an IC50 value of 1.50 µg/mL and a statistically significant difference compared to the negative control (p < 0.001). These results show that the propolis extract from S. postica bees may be a promising alternative for treatment against promastigote forms of L. amazonensis.