Nisin is a low molecular weight bacteriocin which is produced by Lactococcus lactis . Literature about the effects of nisin on pure cultures of rumen bacteria is scarce. The objective of the present study was to investigate the comparative effects of nisin and monensin on pure cultures of some Gram-positive and Gram-negative rumen bacteria. The antibacterial activity assays of nisin and monensin were carried out using broth microdilution method in anaerobic conditions. Antibacterial effect of monensin on Gram-positive rumen bacteria was higher than nisin. Ruminococcus albus and Eubacterium ruminantium were the most sensitive bacteria to monensin. Growth of these bacteria was inhibited completely by monensin, at 6 and 12 µg/mL concentrations respectively. Nisin exhibited stimulatory effects on R. albus , E. ruminantium and, Streptecoccus bovis (p<0.05), unlike monensin. Both nisin and monensin showed potential antibacterial activity on Ruminococcus flavefaciens and Butyrivibrio fibrisolvens (p<0.05) , although they did not inhibit these bacteria completely. Nisin and monensin also did not show inhibitory effect on Methanobacterium formicicum , however the potential antimicrobial activity of monensin on this methanogen was more evident. Gram-negative rumen bacteria, Megasphaera elsdenii and Fibrobacter succinogenes , were resistant to both of nisin and monesin. It was concluded that the different effects of nisin and monensin particularly on some Gram-positive rumen bacteria may be responsible for their inconsistent effects on ruminal fermentation.