Polemonium caeruleum L. is a Chinese medicinal plant used in the treatment of tuberculosis, whooping cough, fever, infertility, epilepsy, endometritis and insomnia. It was also used as sedative and anaesthetic agent. In this work the phytochemical investigation using LC-ESI-QTOF-MS and GC–MS techniques was done for chemical characterization of methanol extracts of underground and aerial plant parts. In the course of our studies on medicinal plants, we evaluated the extracts of P. caeruleum for their biological activities as antimicrobial, antimalarial, anti-leishmanial and antitrypanosomal agents and we investigated their cytotoxicity towards mammalian cells – HEK293 and Vero cell lines, both originating from kidney and also against pharyngeal squamous cell carcinoma, FaDu cell line. LC-ESI-QTOF-MS analysis confirmed the presence of triterpene saponins – oleanane derivatives (polemoniumsaponins, glycosides of theasapogenol derivatives and β-amyrin among others), as well as flavonoid glycosides with most predominant acacetin derivatives. Saponins were more abundant and varied in the extract from underground parts of the plant, while flavonoids dominated in the extract from aerial parts. GC–MS performed after silanization enabled the identification of carbohydrates, fatty acid esters, amino acids and carboxylic acids. In the present study, the antibacterial activity of crude extract of the aerial parts of P. caeruleum against Escherichia coli with an IC50 of 137.0 μg/mL and antitrypanosomal activity of crude extract of the underground parts of P. caeruleum with an IC50 of 16.03 μg/mL were reported. The cytotoxicity of the extract from underground parts of P. caeruleum was in a range 44.5–63.5 μg/ml, while cytotoxicity of the extract from aerial parts ranged from 93.8 to 178.0 μg/ml.