Enterocin CCM 4231 is a bacteriocin with a broad antimicrobial spectrum produced by the ruminal strain Enterococcus faecium CCM 4231. Its inhibitory effect towards enterococci, Ent. faecium EF 26/42, staphylococci, Streptococcus bovis AO 24/85 and Escherichia coli, as well as towards Listeria monocytogenes OHIO strain, in the rumen fluid environment was studied during culture at 37 and 30°C for 24 h and 20 days. Enterocin CCM 4231 was added to the samples at a concentration of 3200 AU ml−1. The best inhibitory effect was noted against enterococci at both cultivation temperatures. A decrease in total cell count from 108 cfu ml−1 to 104 cfu ml−1 and from 105 cfu ml−1 to 101 cfu ml−1 was detected. Addition of enterocin to the rumen fluid also inhibited staphylococci (from 105 cfu ml−1 to 104 cfu ml−1 and/or 103 cfu ml−1). Gram-negative E. coli cells were inhibited at both cultivation temperatures (decrease from 106 cfu ml−1 to 101 cfu ml−1 at 37°C, and from 107 cfu ml−1 to 105 cfu ml−1 at 30°C). Enterococcus faecium EF 26/42 and Streptococcus bovis AO 24/85, the strains growing in the rumen fluid, were the most sensitive to the addition of enterocin during the first 24 h of fermentation (decrease from 1010 cfu ml−1 and 108 cfu ml−1 to 106 cfu ml−1 and 104 cfu ml−1). An antilisterial effect of the bacteriocin was also confirmed. Further application of bacteriocin in ruminal ecology was indicated.