In this study, the essential oil from mustard seed was isolated by simultaneous steam distillation and extraction (SDE) and analyzed by gas chromatography–mass spectrometry (GC–MS). Fourteen components were identified in the mustard seed essential oil with allyl isothiocyanate being the main component (71.06%). The essential oil has a broad-spectrum antimicrobial activity with inhibition zones and MIC values in the range of 9.68–15.57 mm and 128–512 μg/mL respectively. The essential oil was subsequently encapsulated in complex coacervation microcapsules with genipin, a natural water-soluble cross-linker. The optimum parameters for the hardening effectiveness of the genipin-hardened essential oil microcapsules were 8 h at 40 °C and pH 10.0 with a genipin concentration of 0.075 g/g gelatin. The genipin-hardened microcapsules had a particle size of mainly 5–10 μm and strong chemistry stability which is potential for its application in food preservation.