Ruminal bacterial community composition (BCC) and its associations with ruminal fermentation measures were studied in dairy heifers challenged with combinations of grain, fructose, and histidine in a partial factorial study. Holstein-Friesian heifers (n=30) were randomly allocated to 5 triticale grain-based treatment groups: (1) control (no grain), (2) grain [fed at a dry matter intake (DMI) of 1.2% of body weight (BW)], (3) grain (0.8% of BW DMI) + fructose (0.4% of BW DMI), (4) grain (1.2% of BW DMI) + histidine (6g/head), and (5) grain (0.8% of BW DMI) + fructose (0.4% of BW DMI) + histidine (6g/head). Ruminal fluid was collected using a stomach tube 5, 115, and 215min after consumption of the rations and bacterial 16S ribosomal DNA sequence data was analyzed to characterize bacteria. Large variation among heifers and distinct BCC were evident in a between-group constrained principal components analysis. Bacterial composition in the fructose-fed heifers was positively related to total lactate and butyrate concentrations. Bacterial composition was positively associated with ruminal ammonia, valerate, and histamine concentrations in the grain-fed heifers. The predominant phyla were the Firmicutes (57.6% of total recovered sequences), Bacteroidetes (32.0%), and candidate phylum TM7 (4.0%). Prevotella was the dominant genus. In general, grain or histidine or their interactions with time had minimal effects on the relative abundance of bacterial phyla and families. Fructose increased and decreased the relative abundance of the Firmicutes and Proteobacteria phyla over time, respectively, and decreased the abundance of the Prevotellaceae family over time. The relative abundance of the Streptococcaceae and Veillonellaceae families was increased in the fructose-fed heifers and these heifers over time. A total of 31 operational taxonomic units differed among treatment groups in the 3.6h sampling period, Streptococcus bovis was observed in fructose fed animals. The TM7 candidate phylum had an increased abundance of sequence reads by over 2.5 fold due to the introduction of histidine into the diet. Rapid changes in BCC can occur in a short period after a single substrate challenge and the nature of these changes may influence ruminal acidosis risk and differ from those in cattle exposed to substrate challenges over a longer time period.