This study was conducted to determine the effects of Bacillus licheniformis (Bl) and Clostridium butyricum (Cb) and their combinations with yeast culture on in vitro rumen fermentation in a two-way factorial design. Treatments included Bl or Cb at levels of 0, 0.5, 1, 5 and 10 mg and their combination with yeast culture at 0, 18, 27, 36 and 60 mg per 200 mg substrate, respectively. Gas production was recorded after 2, 4, 6, 9, 12, 24, 36, 48 and 72 h incubation. In vitro organic matter digestibility (IVOMD) was estimated by 24 h gas production. Rumen fermentation parameters were determined after 24 h of incubation. Rate constant of gas production was not influenced by Bl or Cb alone, but increased (P<0.05) with inclusion of yeast culture. The IVOMD was influenced (P<0.05) by addition with Bl, Cb or yeast culture, with highest IVOMD observed when Bl or Cb was combined with 60 mg yeast culture. Total volatile fatty acids were affected by Bl and yeast culture (P<0.01), but not by Cb (P>0.05). There were significant interaction effects on pH, acetate to propionate ratio and ammonia-N between yeast culture and Bl or Cb. From the above results, it is indicated that Bl and Cb may be more effective as feed additives when combined with yeast culture than when offered separately.