The composition of the gut microbiota oscillates according to the light–dark cycle. However, the existing literature demonstrates these oscillations only by molecular methods. Microbial cultures are an interesting method for studying metabolically active microorganisms. In this work, we aimed to understand the diurnal oscillation of the intestinal microbiota in Wistar male rats through microbial culture analysis. Over a 24 h period, three animals were euthanized every 6 h. Intestinal segments were dissected immediately after euthanasia and diluted in phosphate-buffered saline (PBS) for plating in different culture media. The CFU/mL counts in feces samples cultured in the Brucella medium were significantly higher at ZT0, followed by ZT6, ZT18, and ZT12 (p = 0.0156), which demonstrated the diurnal oscillation of metabolically active anaerobic bacteria every 6 h using microbial culture. In addition, quantitative differences were demonstrated in anaerobic bacteria and fungi in different gastrointestinal tract tissues.