The present study is the first investigation of the chemical composition, antioxidant, antimicrobial and anti-inflammatory activities of the stem and leaf essential oils from Piper flaviflorum C.DC (SEOP and LEOP), a plant that has been consumed as a wild vegetable, and used as medicine, and spice by the ethnic groups in Xishuangbanna, SW China. Analyzed by GC-MS, 42 and 30 components were identified representing 90.1% and 95.3% of the SEOP and LEOP, with (E)-nerolidol (16.7% and 40.5%), β-caryophyllene (26.6% and 14.6%) and elixene (5.3% and 12.3%) as their main constituents, respectively. Our results indicate that SEOP and LEOP have good anti-inflammatory activity by significantly inhibiting NO production induced by LPS in RAW 264.7 cells at 0.04‰ without effect on cell viability, and negligible antioxidant activity in both ABTS and FRAP assays. Moreover, the LEOP showed comparable activity with the positive control (tigecycline) against Aspergillus fumigatus, with MIC and MBC values ranging from 256 to 1024 μg/mL. The anti-inflammatory activity in LPS-induced RAW 264.7 cells is worthy of further investigation to discover the possible mechanisms of the NO production inhibition effect of these essential oils.