In this study, the antimicrobial and antioxidant properties of yellow gentian roots (Gentiana lutea L.) were investigated. Root samples were collected at the Vranica Mountain, situated in the central part of Bosnia and Herzegovina. The yellow gentian roots were evaluated for antimicrobial activities against four bacteria, namely, Gram-positive bacteria: Staphylococcus epidermidis, Enterococcus faecalis and Gram-negative bacteria: Escherichia coli and Klebsiella pneumoniae. Total antioxidant capacity of root extracts was evaluated using ferric reducing antioxidant power (FRAP) assay. Total phenolic and total flavonoid contents were also evaluated. The antimicrobial assays indicated that the yellow gentian roots were more effective against Staphylococcus epidermidis and Escherichia coli than against Enterococcus faecalis and Klebsiella pneumoniae. The results also showed that the extracts of yellow gentian roots contained relatively high amounts of phenolic compounds and high antioxidant activity rates. The antioxidant activity of the root extracts was found to be positively associated with the total phenolic and flavonoid contents.