Berberine (BER) is a natural alkaloid found in various Berberis species, used traditionally in herbal medicine and dietary supplements. BER exhibited various biological activities, including anti-inflammatory, antioxidant, antidiabetic, and anti-tumour properties by modulating cellular pathways and influencing gene expression. While BER shows promise in various health applications, its possible toxicity raises concerns. In 2023, EFSA issued a call for data on BER- and protoberberine (PROTBER)-containing products, to comprehensively assess its safety and potential health claims. With these premises, new data was collected by assessing: i) the phytochemical profile of BER- and PROTBER-containing preparations by newly developed HPLC-DAD method; ii) in silico and in vitro investigation of pharmacokinetics properties of BER; iii) in vitro cytotoxicity in different human cell lines and, iv) biological and molecular activities of extracts containing BER and PROTBER in in vitro human non-cancer cells. By testing 16 commercial Berberis extracts, the evidence collected showed a good qualitative reliability in terms of phytochemical profile and limited relapses on cytotoxicity even at 200 µg/ml after a 24h of exposure, while evidencing a good cellular bioavailability and cell migration inhibition. The potential for plant-drug interactions affecting CYP450 proved to be minimal. Overall, our work provides a useful overview to better elucidate potential concerns on botanicals containing BER and minor PROTBER commonly used in food supplement.