This study aimed to evaluate the effects of feeding frequency on behavioral patterns and on diurnal fermentation and bacteriome profiles of the rumen and feces in Holstein and Jersey cows. Ten Holstein and 10 Jersey cows were offered a TMR (53:47 forage-to-concentrate ratio dry matter basis) for ad libitum consumption and were randomly allocated within breed to one of the following feeding frequencies: (1) TMR delivered 1×/d (at 0600 h) or (2) TMR delivered 2×/d (at 0600 and 1800 h). The experiment lasted for 28 d with the first 14 d for cow adaptation to the Calan gates and the next 14 d for data collection. On d 23 and 24, an observer manually recorded the time budget (time spent lying, eating, drinking, standing, and milking), rumination activity, and number of visits to the feeding gate from each animal. On d 28, 5 concomitant collections of rumen and fecal samples were performed at intervals of 6 h via esophageal tubing and fecal grab, respectively. The bacteriome composition from these samples was determined through sequencing of the V4 region of the 16S rRNA gene. Feeding frequency did not affect behavioral patterns; however, Holstein cows spend more time lying (15.4 vs. 13.5 ± 0.8 h) and ruminating (401 vs. 331 ± 17.5 min) than Jersey cows. Fermentation profiles were similar by feeding frequency in both breeds. While no major diurnal fluctuations were observed in the fecal bacterial community from both breeds, diurnal fluctuations were identified in the rumen bacterial community from Holstein cows which appeared to follow pH responses. Overall, the bacterial community composition was not differentiated by industry standard feeding frequencies but was differentiated by breed and sample type.