ABSTRACT Citronella (Cymbopogon spp.) essential oil is an important component of various fragrances, cosmetics, and household products. In this study, comparative chemical composition and antimicrobial activity of Java citronella (Cymbopogon winterianus Jowitt ex Bor) essential oil, extracted by different methods, namely hydro-distillation and steam-distillation were determined by GC, GC-MS, and disc diffusion assay and microdilution broth assay. Major constituents of the essential oil were citronellal (2.2â55.4%), geraniol (14.2â53.0%), citronellol (8.2â16.4%), isopulegol (0.3â12.6%), elemol (0.8â8.2%), and limonene (0.2â5.0%). The essential oils showed inhibition of test bacteria with minimum inhibition concentration (MIC) in the range of 250â1000 Âµg/mL and showed anticandidal activity with MIC in the range of 125â500 Âµg/mL. In general, the oils were moderately active against Gram-positive and Gram-negative bacterial strains and also showed good activity against Candidal strains. The essential oils extracted from the herbage by different techniques exhibited variations in their yield, chemical composition, and antimicrobial activities.