Aim This study aimed to develop a simplified chemically defined medium that could sustain the growth and bacteriocin (enterolysin A) production by Enterococcus faecalis B9510. Methods and Results The nutritional requirements of E.faecalis B9510 in a chemically defined medium were determined by single omission experiments. It was observed that eight amino acids (arginine, glycine, histidine, isoleucine, leucine, methionine, tryptophan and valine), three B vitamins (nicotinic acid, Ca-pantothenic acid and pyridoxal) and magnesium sulphate were essential for growth. Based on this information, a Simplified Defined Medium (SDM) was formed consisting of 26 components. Comparison of SDM with M-17 showed that growth and bacteriocin production in SDM was similar to that in M-17. The bacteriocin from SDM was then purified by ultrafiltration. The retentate of ultrafiltration step was analysed by SDS-PAGE and the results showed a single active band in the gel, which was excised and analysed by mass spectrometry, which indicated that the active band was enterolysin A, a cell wall degrading bacteriocin. Conclusions A simplified defined medium can be formulated for the growth and bacteriocin production by Enterococcus faecalis, whose efficiency is comparable with that of a complex commercial medium. Significance and Impact of the Study The development of such a medium can be useful for bacteriocin production and subsequent purification in a simplified manner and, therefore, helpful in the identification of novel bacteriocins.