The qualitative, quantitative and microbial determination of the aqueous extract of comfrey root was done in this paper. The qualitative and quantitative analyses were done by the UHPLC-DAD-HESI-MS method. Allantoin, rosmarinic acid and ellagic acid were identified as major bioactive compounds and their quantification was also done. The obtained results showed a high content of allantoin, ellagic acid and rosmarinic acid (8.91, 7.4 and 12.8%, respectively) which indicated that the comfrey root can be used as a source for the isolation of these three compounds. The results obtained by the determination of the antimicrobial activity showed that Escherichia coli ATCC 8739 and Salmonela typhimirium ATCC 6538 were most sensitive to the aqueous extract of comfrey root. The results showed that allantoin did not express the antimicrobial activity on all the investigated bacteria species, and based on this it can be concluded that allantoin is not responsible for the antimicrobial activity of the aqueous extract of comfrey root.