Spondias pinnata (Linn. f.) Kurz (Anacardiaceae) is widely distributed in tropical Asia, where it is commonly used as a vegetable and fruit, and is attracting increasing research attention. In this study, we investigated the chemical composition and the cytotoxic, antimicrobial, and anti-inflammatory activities of the fruit peel essential oil of S. pinnata (EOSP), which has been consumed as a medicine and condiment in Xishuangbanna, southwest China. A total of 40 components were identified by Gas Chromatography/Mass spectrometry (GC-MS), representing 95.19% of the EOSP, with furfural (17.14%), α-terpineol (13.09%), and ethyl benzoate (9.05%) as the main constituents. EOSP has moderate cytotoxic activity against five cancer cells and obvious antimicrobial activity against five pathogenic strains. In particular, EOSP (Minimal Inhibitory and Fungicidal Concentration, MIC and MFC, 16‒32 µg/mL) showed a 32-times higher inhibition effect against Aspergillus fumigatus than the positive control Tigecycline (MIC and MBC 512‒1024 µg/mL). EOSP also showed strong anti-inflammatory activity by significantly inhibiting nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 cell lines at 0.08‰, with no effect on cell viability. These bioactivities of S. pinnata fruit peel validate its traditional uses and suggest that it could be a new source of natural antimicrobial and anti-inflammatory agents for food or medical industries.