We previously reported that the forest environment enhanced human natural killer (NK) activity, the number of NK cells, and intracellular levels of anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after trips to forests in both male and female subjects. To explore the factors in the forest environment that activated human NK cells, we investigated the effect of essential oils from trees on human immune function both in vitro and in vivo. In the in vitro study, we investigatedthe effect of 8 kinds of phytoncides (wood essential oil) on NK activity and the expression of perforin, granzyme A (GrA) and granulysin (GRN) in human NK cells. We found that phytoncides significantly increased NK activity in a dose-dependent manner and significantly increased the expression of perforin, GrA and GRN. The phytoncides also partially restored NK activity and perforin, GrA and GRN levels reduced by DDVP. We found that pretreatment with phytoncides partially prevented the DDVP-induced inhibition of NK activity. These findings suggest that phytoncides can increase human NK activity. In the in vivo study, twelve healthy male subjects, aged 37-60 years, were put up in urban hotel for 3 nights. Aromatic volatile substances (phytoncides) were produced by vaporizing Chamaecyparis obtusa stem oil with a humidifier in the hotel room during the night. Blood was sampled on the last day and urine was sampled every day during the stay. Similar control measurements were made before the stay on a normal working day. The concentrations of phytoncides in hotel room air were measured. Phytoncide exposure significantly increased NK activity and the numbers of NK, perforin, GRN, and GrA/B-expressing cells, and significantly decreased the concentrations of adrenaline and noradrenaline in urine. These findings indicate that phytoncide exposure and decreased stress hormone levels partially contribute to increased NK activity. © 2013 by Nova Science Publishers, Inc. All rights reserved.