The effect of bromochloromethane-cyclodextrin (BCM-CD) on the microbial community structure in the rumen of steers was examined in this study. Two animals were fed a diet supplemented with BCM-CD (1.0 g/100 kg body weight/d) for two weeks, while two other animals were fed a basal diet during the same experimental period to serve as controls. Two 16S rRNA gene clone libraries were constructed from the rumen fluid of both the control and BCM-treated animals. DNA sequences from the libraries were searched for homology. The statistical differences between both libraries were examined by LIBSHUFF analysis. The DNA sequences of each library were taxonomically classified and the proportion was calculated. A total of 107 (control) and 110 (BCM-treated) clones were analyzed, and the Chao-1 species estimate was 1.6-fold higher in BCM-treated samples. LIBSHUFF comparison showed a statistically significant difference between the two clone libraries. No methanogenic archaeal clone was detected in the BCM-treated clone library. Within Firmicutes, the proportion of Lachnospiraceae clones in the BCM-treated library was 4.5-fold higher than that in the control library. The proportion of Prevotellaceae clones was 1.7-fold higher in the BCM-treated library than in the control library. These results suggest that BCM-CD indirectly affects eubacterial community in the rumen.