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<i>Piper eriopodon</i> is one of the <i>Piper</i> species found in the Sierra Nevada de Santa Marta, and the species has been reported with different compositions of their essential oils (EO). In this study, the volatile fractions/essential oil (by HS-SPME/SDE/MWHD-GC–MS/¹H-NMR) of different parts from the plant were characterized, and assessments of the in vitro bio-properties of the leaf EO were conducted. The results indicated the following: (i) in the volatile fractions were β-caryophyllene (~23%)/myrcene (~20%) (inflorescences) and β-caryophyllene (~43%)/β-selinene (~20%) (leaves) using HS-SPME; myrcene (~31%)/β-pinene (~23%) (inflorescences), gibbilimbol B (~60%) (fruits) and gibbilimbol B (~46%)/β-caryophyllene (~11%) (leaves) through SDE; (ii) leaf EO contained gibbilimbol B (~72%), confirmed with ¹H-NMR; (iii) the cytotoxic values (µg/mL) in erythrocytes/lymphocytes/Hep-2 were HC₅₀: 115 ± 3 (eryth.), LC₅₀: 71 ± 4 (lymph.) and LC₅₀: 33 ± 2 (cell-line); (iv) the antibacterial susceptibilities (ϕ inh. zone, mm; 4–16 µg EO) were 22.5 ± 0.4–97 ± 4 (<i>Staphylococcus aureus</i>), 23 ± 2–77 ± 4 (<i>Escherichia coli</i>) and 17 ± 1–48 ± 3 (<i>Listeria monocytogenes</i>); (v) the TAA value was 2249 ± 130 mmol Trolox^®/kg; (vi) the IC₅₀ value was 13±1 µg/mL (AChE) with 20 ± 0–37 ± 6% repellency (2–4 h, <i>Sitophilus zeamais</i>). Thus, the EO of <i>P. eriopodon</i> leaves from northern Colombia could be a promising species for sustainable exploitation in the future due to its outstanding bioactivities.