Bacopa monnieri (Bmo) is a highly valued medicinal plant, widely used for several pharmacological products in Thailand. The diploid progenitor of Bmo grown in the wild as a natural source has low biomass and bacoside contents. Fifteen treatment combinations between three Bmo explant types (apical shoot, node and leaf segments) and five various colchicine concentrations (0.00, 0.05, 0.075, 0.1, and 0.5% w/v) were determined for a number of polyploid inductions, including bacoside contents and gene expression. Results showed that low concentration (0.05%) of colchicine was more effective in inducing multiple shoots (17.88 regenerants per explant) and tetraploid plantlets (15%) from leaf segments than other treatment combinations. Tetraploid clone (no.4x-3) showed the highest BmoOSC gene expression, bacoside-A3 (4.276 ± 0.019 mg/g dry weight) and bacoside-C contents (5.040 ± 0.078 mg/g dry weight) compared to diploid progenitors and mixoploid plants. These findings indicated that colchicine-induced tetraploids could have beneficial uses for genetic improvements to increase the medicinal value of local Bmo herbal medicine powder production in Thailand.