In this study, lolot pepper (Piper lolot C. DC.), a popular vegetable, a special spice, and effective medicine in Vietnam, was utilized for the extraction of essential oil. Chopped or grounded lolot leaves were hydro-distilled by microwaving supplemented with water levels (200 to 700 mL) in different extraction periods (30 to 120 mins) by different heating power levels (400 to 800 W). The chemical composition of the extract was analyzed by mass spectrometry gas chromatography. Then, the antibacterial and antioxidant properties of lolot essential oil were investigated. As a result, the suitable conditions for the extraction of essential oils of lolot pepper by microwaving were determined by utilizing the chopping raw material with 500 mL distilled water per 500 g of lolot pepper, through a 90-minute extraction at 800 W). The main components of the essential oils of the lolot pepper collected in Dong Thap province were 19 compounds, mainly (E)-nerolidol (18.43%), germacrene D (12.52%), β-elemene (6.7%), βcaryophyllene (4.14%), and α-selinene (4.1%). Lolot pepper essential oils had inhibitory effects on Bacillus subtilis, Escherichia coli, Edwardsiella ictaluri, and Streptococcus pneumoniae. In particular, this essential oil could significantly inhibit E. ictaluri and S. pneumoniae. The antioxidant ability of lolot pepper essential oils tested by the DPPH free radical inhibition activity indicated that the concentration of the essential oils for 50% DPPH inhibition was 16.79 µg/mL.