A polyphenolic extract of Robinia pseudoacacia L. flowers was prepared using a hydro-ethanolic extraction phase. The chemical analysis of the extract consisted in the assessment of the total polyphenolic content (Folin-Ciocalteu method) and the flavonoid fingerprint of the extract determined by high-performance thin-layer chromatography (HPTLC). Two methods ferric reducing antioxidant power (FRAP) and radical scavenging activity with DPPH were used to measure the antioxidant activity of the extract. The cytotoxic and antitumor effects were evaluated on two cell lines: palatal mesenchymal stem cells (pMSCs) and epithelial cells derived from human cervical adenocarcinoma (HeLa (ATTC® CCL-2™)). The chemical content of Robinia pseudoacacia L. flowers extract reveals the presence of apigenin-7-glucoside and rutin. Results indicated high antioxidant activity with both methods (FRAP and DPPH). No cytotoxic effects of the extract were observed on the pMSCs cell line. The extract induced significant necrosis and apoptosis of the HeLa cells, proving an antitumor effect in vitro. The data confirm the antioxidant and antitumor effects of the polyphenols and the therapeutic potential of Robinia pseudoacaia flowers extract. In the last years, the therapeutic effect of the compounds from Robinia pseudoacacia flowers is less discussed; even the traditional medicine mentions them as helpful medicinal means. The present paper points out the correlation between Robinia pseudoacacia flowers polyphenolic compounds and their chemical and biological effects.