Hernandulcin (HE) is a non‐caloric sweetener synthesized by the Mexican medicinal plant Phyla scaberrima. Herein we present the results of HE production through cell suspensions of P. scaberrima as well as the influence of pH, temperature, biosynthetic precursors and potential elicitors to enhance HE accumulation. The incorporation of mevalonolactone (30–400 mg L−1) farnesol (30–400 mg L−1), AgNO3 (0.025–0.175 M), cellulase (5–60 mg L−1; 0.3 units/mg), chitin (20–140 mg L−1) and (+)‐epi‐α‐bisabolol (300‐210 mg L−1) to the cell suspensions, resulted in a differential accumulation of HE and biomass. Among elicitors assayed, chitin, cellulase and farnesol increased HE production from 93.2 to ∼160 mg L−1 but, (+)‐epi‐α‐bisabolol (obtained by a synthetic biology approach) increased HE accumulation up to 182.7 mg L−1. HE produced by the cell suspensions was evaluated against nine strains from six species of gastrointestinal bacteria revealing moderate antibacterial activity (MIC, 214–465 μg mL−1) against Staphylococcus aureus, Escherichia coli and Helicobacter pylori. Similarly, HE showed weak toxicity against Lactobacillus sp. and Bifidobacterium bifidum (>1 mg mL−1), suggesting a selective antimicrobial activity on some species of gut microbiota. According to our results, chitin and (+)‐epi‐α‐bisabolol were the most effective molecules to enhance HE accumulation in cell suspensions of P. scaberrima.

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