This research aimed to apply bacteriocin producing strain Bacillus subtilis (P5-6) as protective culture against Staphylococcus aureus in bamboo shoot pickle. Expression of genes associated with bacteriocin activity of the B. subtilis (P5-6) was investigated. Only subtilin gene expressions were observed along with housekeeping gene BA-rpoB when co-cultured with S. aureus. B. subtilis (P5-6) protective culture was made in a simple form as dried mango pieces containing 2.5 mg/g salt with viable count at 6.25 log CFU/g and water activity (aw) at 0.49, respectively. The Protective culture was made by immobilizing cells and culture of P5-6 in freeze-dried mango pieces and drying with two-step drying process.The Minimum Inhibitory Concentration (MIC) of the protective culture was 20 AU/mg and Minimum Bactericidal Concentration (MBC) at 80 AU/mg. When applied as bio-preservative in bamboo shoot pickle, the protective culture significantly (p<0.05) reduced the level of S. aureus contamination and had no impact on this food properties. This study demonstrated the potential of P5-6 in this simple protective culture form in controlling the growth of S. aureus, therefore it could be a potential for further development as bio-preservative to enhanced safety of food products.