This study investigated the seasonal variation of the chemical composition and antimicrobial activity of Eucalyptus radiata leaf essential oil. Young and mature Eucalyptus radiata leaf material was collected monthly (January 2014 to December 2014), hydrodistilled and analyzed using GC-MS. Essential oil yields ranged from 0.14% to 4.31% (w/w). The major compounds were 1,8-cineole (65.7% ± 9.5), α-terpineol (12.8% ± 4.4) and limonene (6.5% ± 2.4). Chemometric tools were used to determine seasonal variations, which showed slight variance in E. radiata chemistry between seasons. The minimum inhibitory concentration (MIC) assay showed that the highest activity was noted against the Streptococci (0.19â2.00 mg/mL) and Lactobacillus acidophilus (0.19â1.75 mg/mL). The activity of the E. radiata leaf essential oil is dependent on the unique ratio of its compounds. The E. radiata leaf essential oil showed good oil yields, a relatively consistent chemical profile and noteworthy antimicrobial activity that rivals other commercial Eucalypt counterparts.