Essential oils are mixtures of different volatile aromatic compounds extracted from plants. Since the discovery of their antifungal and antimicrobial properties, preparations of essential oils have been applied in pharmacology, medical microbiology, phytopathology and food preservation. Nowadays, the problem of environmental pollution and ecological environment destruction has been more and more serious. The study of how to utilize the plant resources' environmental effect has become an important field of the plant-resources utilization, and it also has been an important subject in academic field. With the increasing public concern on the level of their effects about environment, Fortunella calamondin Rutaceaei was used as an example by examining its anti-bacterial and anti-tumor activity in this paper. As the oil’s antifungal activity is a basic research work for the application of its environmental functions of essential oils, the study concerning the composition of essential oil from Fortunella calamondin is very limited. At first the volatile oil form its leaves was obtained by steam distillation, then its chemical constituents were analyzed by GC-MS. After that biological activity of the volatile oil was screened with microbiological method and Alamar Blue method. With the analysis of its constituents and biological activity of volatile oil from the leaves of the Fortunella calamondin, 23 compounds were identified, showing 99.578% of the total contents; the tested Escherichia coli, Staphylococcus aureus and Rhodotorula glutinis showed great sensitivity to the volatile oil, and the volatile oil could also obviously inhibit the growth of cultured tumor cells. And the inhibitory effects on the cell viability were obviously dose-dependent with a inhibition rate of 82.56% at a volatile oil concentration of 0.1g/L.