Propolis was shown to exert antimicrobial, antioxidant, anti-inflammatory, and anticancer activities. Its composition is influenced by seasonal, climatic and phytogeographic conditions. Further variability derives from the extraction methods. Multi Dynamic Extraction Method (MED) has been recently proposed to improve extracts reproducibility. Here, the cytotoxic/anticancer activity of three MED extracts of poplar-type propolis was assayed on human promyelocytic leukaemia HL60, human monocytic leukaemia THP-1, human osteosarcoma MG63, murine fibroblast L929 and human mesenchymal cells (hMSCs). As far as we are aware of, MG63 cells have never been challenged with propolis before, while few studies have so far addressed the effects of propolis on non-tumor cell lines. Consistent results were observed for all propolis preparations. The extracts turned out mildly cytotoxic toward cancer cells, in particular osteosarcoma cells (IC50: 81.9–86.7 µg/ml). Nonetheless, cytotoxicity was observed also in non-tumor L929 cells, with an even lower IC50. hMSCs demonstrated the lowest sensitivity to propolis (IC50: 258.3–287.2 µg/ml). In THP-1 cells, extracts were found to stimulate apoptosis caspase 3/7 activity. The IC50 values observed with osteosarcoma and leukaemia cells do not support a relevant cytotoxicity (as the figures abundantly exceeded 30 µg/ml), despites some selective activity exhibited with HL60 cells. The results confirm the validity of the extraction method, emphasizing the need to assess the selectivity of the interaction with cancer cells when screening for anticancer-drug candidates.