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Propolis is one of the most widely used products in traditional medicine. One of the most prominent types of Brazilian propolis is the red one, whose primary botanical source is Dalbergia ecastaphyllum(L.) Taub. Despite the potential of Brazilian red propolis for developing new products with pharmacological activity, few studies guarantee safety in its use. The objective of this study was the evaluation of the possible toxic effects of Brazilian red propolis and D. ecastaphyllum, as well as the cytotoxicity assessment of the main compounds of red propolis on tumoral cell lines. Hydroalcoholic extracts of the Brazilian red propolis (BRPE) and D. ecastaphyllumstems (DSE) and leaves (DLE) were prepared and chromatographed for isolation of the major compounds. RP-HPLC-DAD was used to quantify the major compounds in the obtained extracts. The XTT assay was used to evaluate the cytotoxic activity of the extracts in the human fibroblast cell line (GM07492A). The results revealed IC50values of 102.7, 143.4, and 253.1 μg/mL for BRPE, DSE, and DLE, respectively. The extracts were also evaluated for their genotoxic potential in the micronucleus assay in Chinese hamster lung fibroblasts cells (V79), showing the absence of genotoxicity. The BRPE was investigated for its potential in vivotoxicity in the zebrafish model. Concentrations of 0.8–6.3 mg/L were safe for the animals, with a LC50of 9.37 mg/L. Of the 11 compounds isolated from BRPE, medicarpin showed a selective cytotoxic effect against the HeLa cell line. These are the initial steps to determine the toxicological potential of Brazilian red propolis.