This study explored whether aromatherapy increases the parasympathetic component (root mean square of successive differences [RMSSD]) of heart rate variability (HRV) in horses. Horses are highly sensitive, easily stressed, have an acute sense of smell, and have been shown to benefit from aromatherapy. The protocol, performed on eight dressage horses, followed a crossover design with an experimental treatment of humidified essential oil (lavender, Lavandula angustifolia) and a control treatment of humidified air. Heart rate variability was recorded for 7 minutes before, during, and immediately after treatment, and 30 minutes after treatment. The protocol was repeated with chamomile, (Matricaria recutita). Lavender transiently increased RMSSD from 86.5 ± 14.2 ms (standard error of the mean [SEM]) to 104.3 ± 14.3 ms (P = .020) and reduced percentage of very low frequency HRV oscillations from 81.2% ± 3.9 (SEM) to 72.1% ± 7.9 (P = .039) immediately after treatment. Chamomile had variable effects, none of which reached significance. These results indicate that lavender is effective as a calming agent for horses.