Piper nigrum (Black pepper), popularly called the "King of Spices" is one of nature's most ancient and miraculous traditional plants to treat several deadly maladies. The essential oil of P. nigrum is highly considered to have illustrious therapeutic significance in domestic and industrial applications. To validate the traditional claim of its essential oil's efficacy, the proposed study was designed to analyze the volatile profile and assess the antimicrobial and antiproliferative effect of P. nigrum fruit essential oil. This is the first study on the analysis of essential oil of P. nigrum collected from Eastern India. The Gas chromatography-mass spectrometry analysis of P. nigrum essential oil revealed beta-caryophyllene (25.6%), limonene (15.7%), sabinene (11.6%), beta-pinene (11.1%) and delta-carene (8.6%) as its major constituents. In antibacterial assays, P. nigrum essential exhibited significant activity against multiple microbial pathogens like Salmonella enterica serovar Typhi (ZOI-19 +/- 0.4 mm; MIC-0.781 mu g/ml), Escherichia coli (ZOI-18 +/- 0.4 mm; MIC-1.562 mu g/ml) and Staphylococcus saprophyticus (ZOI-18 +/- 0.4 mm; MIC-1.562 mu g/ml). MTT assay demonstrated selective cytotoxicity against cancerous cells such as HepG-2 (IC50-71.2 +/- 1.3 mu g/ml); PC-3 (IC50-68.38 +/- 1.2 mu g/ml) as compared to noncancerous 3T3L-1 cells (IC50- 235.77 +/- 1.6 mu g/ml).To the best of our knowledge, this is the first-ever study from India on the cytotoxic potential of P. nigrum essential oil against the above two robust cancerous cell lines. The present findings revealed that P. nigrum essential oil exhibited remarkable therapeutic potential, which could enhance its medicinal worth and commercial significance in current scenario and in future perspectives for designing antimicrobial and anticancer drugs.