Background The effect of polyamines (PAs) along with cytokinins (TDZ and BAP) and auxin (IBA) was induced by the multiple shoot regeneration from leaf explants of gherkin (Cucumis anguria L.). The polyphenolic content, antioxidant and antibacterial potential were studied from in vitro regenerated and in vivo plants. Results Murashige and Skoog (MS) medium supplemented with 3% sucrose containing a combination of 3.0 µM TDZ, 1.0 µM IBA and 75 µM spermidine induced maximum number of shoots (45 shoots per explant) was achieved. Regenerated shoots elongated in shoot elongation medium containing 1.5 µM GA3 and 50 µM spermine. The well-developed shoots were transferred to root induction medium containing 1.0 µM IBA and 50 µM putrescine. Rooted plants were hardened and successfully established in soil with a 95% survival rate. Twenty-five phenolic compounds were identified by ultra-performance liquid chromatography (UPLC) analysis The individual polyphenolic compounds, total phenolic and flavonoid contents, antioxidant and antibacterial potential were significantly higher with in vitro regenerated plants than in vivo plants. Conclusions Plant growth regulators (PGRs) and PAs had a significant effect on in vitro plant regeneration and also a biochemical accumulation of flavonols, hydroxybenzoic and hydroxycinnamic acid derivatives in C. anguria. Due to these metabolic variations, the antioxidant and antibacterial activities were increased with in vitro regenerated plants than in vivo plants. This is the first report describing the production of phenolic compounds and biological activities from in vitro and in vivo regenerated plants of C. anguria.