Previous studies from our laboratory showed that several non-specific, polysaccharide-producing soil bacteria could be effectively used to prevent hyperhydricity. In this study, prevention of hyperhydricity of shoot cultures of oregano clonal line O-1 was investigated over multiple subculture cycles without re-inoculation of shoots. Results clearly indicate that hyperhydricity was prevented in oregano shoot cultures over eight subculture cycles without any re-inoculation after the initial inoculation with several polysaccharide-producing bacteria in the first cycle. Hyperhydricity-related parameters of subculture cycles 7 and 8, reported in this manuscript, showed that the water content was significantly reduced in response to all bacteria tested. The total phenolics content, on a fresh-weight and dry-weight basis, was stimulated significantly by all bacterial treatments except Pseudomonas stutzeri in the seventh cycle. The chlorophyll content was significantly stimulated in all treatments on a fresh-weight basis in the two subculture cycles. On a dry-weight basis, except for P. stutzeri, the bacterial species tested induced significant increases in chlorophyll content. Plant growth in response to all bacteria was reduced. In spite of growth reduction, the numbers of shoot nodes available for propagation were not reduced and all the shoot tissues were unhyperhydrated. These results also suggested that prevention of bacteria-mediated hyperhydricity may be linked to survival of bacteria in the stem, which may be carried through to the next subculture. Acclimation studies showed that bacteria-containing shoots performed better and further strengthened the case for use of non-pathogenic, polysaccharide-producing bacteria for hyperhydricity control in commercial plant tissue cultures.