Piperaceae family includes numerous species used for food, spices, and medicinal purposes. In this study, the chemical composition and bioactivities (antioxidant, anticholinesterase, and anti-inflammatory activities) of the essential oil from Piper rostratum Roxb. were investigated. The essential oil was extracted via hydrodistillation and thoroughly analysed using gas chromatography-flame ionisation detection (GC-FID) and gas chromatography-mass spectrometry (GC-MS). A total of 20 chemical components were identified, accounting for 94.2% of the total oil. The major components were γ-muurolene (14.1%), δ-cadinene (13.2%), allylpyrocatechol diacetate (11.5%), chavicol (8.2%), α-humulene (7.8%), and hydroxychavicol (6.9%). The antioxidant activity was evaluated using the DPPH free radical scavenging assay, the acetylcholinesterase inhibitory activity was measured using the Ellman method, and the anti-inflammatory activity was assessed through lipoxygenase enzyme inhibition. The essential oil demonstrated moderate activity in DPPH free radical scavenging (IC50 = 98.5 µg/mL), acetylcholinesterase inhibition (IC50 = 89.2 µg/mL), and lipoxygenase inhibition (IC50 = 77.2 µg/mL).