The goal of the studies was to check if biomass from in vitro cultures of certain Eryngium species could be a source of rosmarinic acid glucoside, which is expected to be more convenient for use in formulations than rosmarinic acid, a compound known for its many biological properties and valuable in medicinal and cosmetological applications. Four positional isomers of β-glucoside of rosmarinic acid are known in the plant kingdom. The investigations were conducted on in vitro cultures of E. planum L., E. campestre L. and E. maritimum L. Using chromatography on synthetic polymers and structural elucidation by spectral methods, like mass spectrometry (HR-ESI-MS, HR-ESI-MS/MS) and nuclear magnetic resonance (1D and 2D NMR), rosmarinic acid 4′-O-β-glucopyranoside (RAG4`) (1) (glucose linked to dihydroxyphenyllactic acid residue) and rosmarinic acid (RA) (2) were isolated and identified from callus cultures of E. planum and root cultures of E. planum and E. campestre. Ambiguity regarding the appropriate establishment of a glucosidic linkage site in the rosmarinic acid molecule has been discussed. The isolation yields of RAG4′ were 0.375 mg/g and 0.639 mg/g dry biomass weight and were higher than that reported for the roots of the soil-grown Eryngium plant. However, it was lower than the yield of RA – 2.500 mg/g. Based on the reported anti-inflammatory effects of RA and RAG4` potential anti-tumor effects have been evaluated on five human carcinoma cell lines (MCF-7, MDA-MB-231, HT-29, Caco-2, OVCAR-3) and one non-tumorigenic mammary epithelial cell line (MCF-12A) using MTT (3-(4,5-dimethylthiazol-2-yl)− 2,5-diphenyltetrazolium bromide) assay. The highest cytotoxic activity of RAG4` with IC50 401.2 ± 3.41 µM was shown against the Caco-2 cell line. However, RA exhibited significantly higher overall antiproliferative/cytotoxic activity against all cell lines used in the experiment than its derivative. Worth mentioning is that RAG4′ showed no effect on the non-tumorigenic MCF-12A cell line, indicating that it could be safe for use in pharmaceutical and cosmetic formulations. The presented study is the first report on the isolation of rosmarinic acid 4′-O-β-glucopyranoside from biomass produced by means of plant biotechnology, as well as the investigation of its cytotoxic activity. Display omitted •rosmarinic acid in a glucoside form will be more convenient for use.•in vitro cultures of Eryngium are a source of rosmarinic acid 4′-O-β-glucopyranoside.•spectral analysis is necessary for distinction of glucosidic linkage sites.•rosmarinic acid 4′-O-β-glucopyranoside has lower cytotoxicity than rosmarinic acid.