Four surface sterilization agents and two hot-water treatments were evaluated with a view to eliminating bacterial contamination in sugarcane (Saccharum hybrids) buds used for in vitro initiation. None of the treatments achieved total elimination of contaminants. Disinfection in either a 2.7% solution of sodium hypochlorite (pH 6) or 1% mercuric chloride were equally good, resulting in 46.8% and 56.3% clean cultures respectively. However, sterilization with 2.7% sodium hypochlorite (pH 6) was most phytotoxic and 100% tissue necrosis and bud death were recorded. The Australian cold-soak hot-water treatment (consisting of a 48-h soak in running water followed by hot-water treatment at 50 °C for 3 h on the following day) reduced contamination from 70% to 25%. With the dual hot-water treatment (setts immersed in water at 52 °C for 20 min and then at 50 °C for 3 h on the following day) a decrease to 45% was observed. Bacterial contaminants were identified by fatty acid profiling and were found to belong mainly either to the Enterobacteriaceae family or the genus Bacillus.