<p><i>Piper barberi</i> Gamble is endemic to tropical wet evergreen forests of the Southern Western Ghats and is an endangered dioecious climber belonging to the family Piperaceae. It is distinct from other South-Indian species of Piper, in morphology. Concerning conservation and medicinal interest, the current study focuses on the standardization of a novel in vitro propagation protocol by eradication of endophytic fungi from nodal and shoot tip explants, using Tagstin and Plant Preservative Mixture (PPM) and successive multiple shoot induction using AgNO₃. The explants inoculated in Murashige and Skoog (MS) medium in different strengths, supplemented with varying concentrations of PGRs and their combinations to establish a robust micropropagation. The highest percentage of shoot induction 93.33 ± 5.77, for nodal explant was observed in half-strength MS medium augmented with BA 0.5 mgL⁻¹ + IBA 1 mgL⁻¹. Nodal explants inoculated in the same shooting medium supplemented with different concentrations of AgNO₃ (0.5–3.0 mgL⁻¹) induced multiple shooting, and the highest shoots per explant ratio (17.33 ± 3.79) was observed in medium supplemented with 1.0 mgL⁻¹ AgNO₃. In vitro regeneration of roots in quarter-strength MS medium with 2 mgL⁻¹ IBA resulted in an average of 6.33 ± 1.53 roots per explant. Genetic fidelity profiling of regenerated plants using ISSR primers resulted in an acceptable monomorphism in banding, without any variation. The current study suggests an in vitro conservation strategy for the declining species <i>P. barberi</i> by proposing a novel and cost-effective sterilization protocol, and provisions to procure abundant and stable resources for commercial and pharmacological studies.</p> Graphical abstract <p></p>