The presence of antimicrobial substances at low concentration may act as chemical signals in altering metabolic processes that take place in bacteria. The chemical signals may activate lots of signal transduction that result in the secretion of bioactive proteins by the stimulated bacterial cells. The bioactive proteins production is strongly influenced by certain factors such as pH and nutrient sources. Therefore, the study of environmental stress factors and growth conditions is very important in inducing the production of bioactive proteins by microbes. The main objective of this study is to evaluate the effects of environmental stress factors and growth conditions on the production of bioactive proteins by Bacillus subtilis ATCC21332 in the presence of Cymbopogon flexuosus essential oil. The microbial proteins were produced by fermentation process using Nutrient broth with adjustment of pH media (pH 6, 7 and 8), and supplementation with 1% (w/w) of three different nutrients, including carbon sources (glucose, sucrose and starch), nitrogen sources (urea, casein and gelatin) and inorganic salts (calcium chloride, sodium nitrate, sodium dihydrogen phosphate) separately. The extracellular protein produced by B. subtilis ATCC21332 was isolated and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to detect appearance of new protein bands. Results showed that new extracellular proteins were synthesized by B. subtilis ATCC21332 after being induced with C. flexuosus essential oil and grown in each media with 1% (w/w) of starch, 1% (w/w) of gelatin, 1% (w/w) of casein, 1% (w/w) of sodium dihydrogen phosphate and 1% (w/w) of calcium chloride with approximate size of 30.43 kDa, 30.66 kDa, 31.92 kDa, 31.35 kDa and 30.80 kDa respectively. Hence, B. subtilis ATCC21332 in the presence of 0.01 MIC C. flexuosus essential oil under different conditions are able to produce or secrete new extracellular proteins. � 2018, Malaysian Society of Applied Biology. All rights reserved.