Zanthoxylum motuoenseHuang, native to Tibet, China, is a newly discovered Chinese prickly ash, which, recently, has increasingly attracted the attention of researchers. In order to understand its volatile oil compositions and flavor characteristics, and to explore the flavor difference betweenZ. motuoenseand the common Chinese prickly ash sold in the market, we analyzed the essential oils ofZ. motuoensepericarp (MEO) using HS-SPME/GC×GC-TOFMS coupled with multivariate data and flavoromics analyses. The common commercial Chinese prickly ash in Asia,Zanthoxylum bungeanum(BEO), was used as a reference. A total of 212 aroma compounds from the 2 species were identified, among which alcohols, terpenoids, esters, aldehydes, and ketones were the major compounds. The predominant components detected from MEO were citronellal, (+)-citronellal, and β-phellandrene. Six components—citronellal, (E,Z)-3,6-nonadien-1-ol, allyl methallyl ether, isopulegol, 3,7-dimethyl-6-octen-1-ol acetate, and 3,7-dimethyl-(R)-6-octen-1-ol—could be used as the potential biomarkers of MEO. The flavoromics analysis showed that MEO and BEO were significantly different in aroma note types. Furthermore, the content differences of several numb taste components in two kinds of prickly ash were quantitatively analyzed using RP-HPLC. The antimicrobial activities of MEO and BEO against four bacterial strains and nine plant pathogenic fungi were determined in vitro. The results indicated that MEO had significantly higher inhibitory activities against most microbial strains than BEO. This study has revealed the fundamental data in respect of the volatile compound properties and antimicrobial activity ofZ. motuoense, offering basic information on valuable natural sources that can be utilized in the condiment, perfume, and antimicrobial sectors.