The essential oil of Peucedanum dhana A. Ham growing in Thailand was investigated for their volatile compounds and screened for its antimicrobial, antioxidant, and cytotoxic activities. Forty-two compounds were identified, with the most important compounds being trans-piperitol, o-cymene, β-pinene, γ-terpinene, and limonene. Essential oil of P. dhana has remarkable antimicrobial activity against tested pathogens with minimum inhibitory concentration and minimum microbicidal concentration values, ranging from 62.50–250 µg/mL and 250-1,000 µg/mL, respectively. The antioxidant activity of P. dhana oil was measured using DPPH and ABTS scavenging activity assays. The IC50 values using the DPPH and ABTS method were 9.13 and 9.36 mg/mL, respectively. The total phenolic compounds in P. dhana oil was 183.05 mg GAE/100 g dry weight. The cytotoxic effect of P. dhana oil was tested against Hela, A549, SW480, and 3T3L1 cells. The essential oil had cytotoxicity against all cancer cells, with significant cytotoxicity towards the SW480 cell. The results indicate that the P. dhana essential oil could be used as a source of functional ingredients in food and pharmaceutical applications.