Neutral cellulase-assisted steam distillation (NCSD) was developed to extract essential oil from Valeriana officinalis L. root, and a single-factor experiment was designed to optimize the NCSD. Chemical composition was subsequently compared between Valeriana officinalis L. root essential oil (VORO) extracted by NCSD and conventional steam distillation (SD). Results show that the highest yield of VORO extracted by NCSD was 68.09% higher than that extracted by SD. Results of antibacterial activity indicate that the two VOROs had an equally strong antibacterial activity of Bacillus subtilis, Staphylococcus aureus, and Escherichia coli with minimum inhibitory concentration (MIC) of 0.88, 1.75, and 3.50 mg/mL, respectively. Both VOROs also showed sufficiently high antioxidant and anti-dust mite abilities. The VORO concentration higher than 0.35 mg/mL was regarded as an A-grade dust mite repellent and B-grade miticide. This study has emphasized the significances of VORO in the food and textile industries as a potential additive.