Leishmaniasis is caused by protozoans of the genus Leishmania, and its treatment is highly toxic, leading to treatment discontinuation and the emergence of resistant strains. In this study, we assessed the leishmanicidal activity and chemical composition of red propolis collected from the Amazon-dominated region of northern Tocantins State, Brazil. The MTT assay was employed to determine the samples' activity against Leishmania amazonensis promastigotes and their cytotoxicity against RAW macrophages. Spectrophotometric assays were utilised to measure the concentrations of total phenolics and flavonoids, while high-performance liquid chromatography coupled to a mass spectrometer (LC-MS/MS) was used to determine the chemical composition. An in silico study was conducted to evaluate which compounds from Brazilian Amazon red propolis may correlate with this biological activity. Brazilian Amazon red propolis exhibited a high concentration of phenolic compounds and an inhibitory activity against L. amazonensis, with an IC50 ranging from 23.37 to 36.10 µg/mL. Moreover, fractionation of the propolis yielded a fraction with enhanced bioactivity (16.11 µg/mL). Interestingly, neither the propolis nor its most active fraction showed cytotoxicity towards macrophages at concentrations up to 200 µg/mL. The red colour and the presence of isoflavonoid components (isoflavones, isoflavans, and pterocarpans) confirm that the substance is Brazilian red propolis. However, the absence of polyprenylated benzophenones suggests that this is a new variety of Brazilian red propolis. The in silico study performed with two of the main leishmanicidal drug targets using all compounds identified in Amazon red propolis reported that liquiritigenin was the compound that exhibited the best electronic interaction parameters, which was confirmed in an assay with promastigotes using a standard. The findings indicate that Amazon red propolis possesses leishmanicidal activity, low toxicity, and significant biotechnological potential.