Anubias barteri is an ornamental aquatic plant of economic importance worldwide. Effective removal of contaminants has been a major problem for the in vitro propagation of A. barteri. Hydrogen peroxide (H2O2) was applied as a pre-disinfectant to treat rhizome bud explants of A. barteri var. nana ‘Mini’ followed by disinfection with mercuric chloride (HgCl2) to eliminate in vitro contamination. However, application of 0.1% HgCl2 for 5 min without pre-disinfecting with H2O2 significantly reduced the contamination rate to 44.4% compared to other combinations. Moreover, in vitro shoots of A. barteri var. nana ‘Mini’ were used as plant materials for determining the basal requirement of inorganic minerals. Optimal plant growth was achieved on 1/2 Murashige and Skoog (MS) basal medium. Rhizome bud explants were cultured on 1/2 MS basal medium supplemented with 6-benzyaminopurine (BAP) or thidiazuron (TDZ) in combination with α-napthaleneacetic acid (NAA) to screen for the optimal combinations for shoot proliferation. BAP or TDZ alone at 1 mg L-1, and the combination of 0.5 mg L-1 TDZ and 1 mg L-1 NAA showed significantly higher shoot proliferation rate ranged from 2.4 to 2.7-fold after being cultured for 5 weeks. Ex vitro plantlets after acclimatization adapted well in aquarium. Successful plant regeneration of A. barteri var. nana ‘Mini’ was established through direct shoot organogenesis from rhizome buds and could be used for mass propagation.