The present study attempted to evaluate the suitable conditions for essential oil extraction from the fruit of Triphasia trifolia using hydrodistillation technique. The effect of various factors such as material size, solvent to solid ratio, heating temperature and distillation time, state of maturation, and fruit preservation on the oil yield was investigated. The physicochemical property, chemical composition, and biological properties were accessed. The highest yield of essential oil (7.9 mL/kg dry matter) from T. trifolia was obtained at optimal conditions such as fine particle size, solid to solvent ratio of 1:5, temperature oil bath of 140 °C, and distillation time of 3 h. The state of maturation of the fruits significantly affects the essential oil yield and insignificantly influences chemical composition based on the gas chromatography-mass spectrometry (GC-MS) analysis. It was found that the essential oil yield decreases significantly when ripened fruits are not kept in cool conditions before distillation. It implied that ripened fruits should be immediately processed or kept in cool storage to maintain the essential oil yield. Our findings will help produce high-quality essential oil from T. trifolia at a pilot or industrial scale.