This study was conducted to examine the effects of clay (CL) and Saccharomyces cerevisiae fermentation product (SCFP) sequestering agents on the ruminal bacterial community of Holstein dairy cows challenged with aflatoxin B₁ (AFB₁). Eight lactating cows stratified by milk yield and parity were randomly assigned to 1 of 4 treatments in a 4 × 4 Latin square with 2 replicate squares, four 33-d periods and 5-d washout between periods. Treatments were (1) Control (basal diet); (2) T (Control + 63.4 µg/kg AFB₁); (3) CL (T + 200 g/head/d sodium bentonite clay, top-dressed); (4) CL+SCFP (CL +19 g Diamond V NutriTek + 16 g MetaShield, top-dressed). During each period, cows were adapted to the basal diet (36.1% corn silage, 8.3% alfalfa hay and 55.6% concentrate; DM basis) from d 1 to 25 (pre-dosing period), orally dosed with AFB₁ from d 26 to 30 (dosing period), followed by toxin withdrawal from d 31 to 33 (withdrawal period). Data were analyzed using GLIMMIX procedure of SAS. During the pre-dosing period, feeding CL+SCFP increased the relative abundance of Prevotella compared with the Control (43.6 vs. 40.0%; P = 0.02). During the dosing period, feeding T had no effects on the ruminal bacterial community (P > 0.10) but CL+SCFP increased the abundance of Fibrobacter compared with T and Control (1.45 vs. 0.97 and 1.05%; P = 0.04 and 0.10, respectively). Ruminal fermentation parameters did not differ across treatments. Feed efficiency was positively correlated (r >0.30, P < 0.05) with relative abundance of Treponema, and Coprococcus. Yields of milk and milk components correlated positively (r >0.42; P < 0.01) with relative abundance of unclassified Succinivibrionaceae, or Coprococcus, suggesting they play an important role in increasing milk production. The ruminal microbial community was not affected by T or CL but CL+SCFP increased the abundances of Prevotela and Fibrobacter in the predosing and toxin-dosing periods, perhaps resulting in the increased milk yield observed in our companion study.