The ability to calm horses affects both the safety and well-being of human-horse relationships. However, not many natural calmatives are known, nor the efficacy of these in supporting a state of calmness in horses. This study used both qualitative and quantitative methods to test whether aromatherapy has a measurable and calming effect on equine facial expression and a range of physiological parameters. Nine horses of different ages, genders and breeds were included in a crossover design with humidified essential oils to determine the calming effect of vetiver (Vetiveria zizanioides), spikenard (Nardostachys jatamansi) and roman chamomile (Anthemis nobilis) in relation to water (negative control) and lavender oil (positive control) in horses standing still. The results revealed significantly lowered heart rate (P < 0.05 - <0.001) and respiratory tidal volume (P < 0.05 - <0.001) for the tested essential oils, while spikenard oil was best at inducing a relaxed facial expression (60% of the full-time scale for 8 horses; 75% of the full-time scale for 7 horses) and roman chamomile oil was statistically most successful at reducing the incidence of spontaneous muscle contractures in M. Temporalis (P < 0.001) and M. Cleidomastoideus (P < 0.05) compared to the other aromatherapy treatments. It is concluded that aromatherapy has both qualitative and quantitative effects on physiological parameters in static horses, and moreover, that essential oils appear to have a calming effect.