Eucalyptus benthamii Maiden et Cambage is a medicinal plant that is also widely used for reforestation in Southern Brazil. However, restricted data about its essential oil are available. The anatomical analysis was performed by the usual light and scanning microtechniques. The identification of volatile constituents was achieved by GC-MS and GC-FID. The antioxidant potential was evaluated by the phosphomolybdenum and 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) methods. In vitro antimicrobial activity was investigated by the agar disk diffusion technique. Fumigant and repellent activities were carried out against head lice. Secretory ducts containing volatile compounds were observed in mesophyll and petiole. The major volatile components were α-pinene (22.6%), γ-terpinene (16.6%), p-cymene (10.6%), aromadendrene (9.9%), and terpinen-4-ol (5.9%). For phosphomolybdenum method, the volatile oil of E. benthamii showed a total antioxidant activity (43.15 ± 1.08%) higher than that achieved for rutin (20.70 ± 1.69%). However, a low antioxidant potential was observed by DPPH method (IC50= 3209.66 ± 218.86 µg.mL-1). The essential oil of E. benthamii had an antimicrobial effect against Candida albicans, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pyogenes strains. In the insecticidal activity against head lice, the investigated volatile oil demonstrated promising fumigant (Knockdown time50= 36.67 ± 2.89 min) and repellent (Repellent index = 35.03 ± 13.13%) effects. This study was supported by Fundação Araucária (Brazil).