The influence of slow-release urea ( urea-calcium sulphate mixture; U-CaS) in feed blocks on rumen micro-organisms, predominant cellulolytic bacteria, microbial protein synthesis and ecology was studied in Thai native beef cattle. Four animals with an initial body weight of 100 ± 3.0 kg were randomly assigned to a 4 × 4 Latin square design with four dietary treatments (U-CaS in iso-nitrogen feed blocks at 0, 120, 150 and 180 g/kg dry matter (DM), respectively; U-CaS replaced urea). After 21 days of experimental feeding, rumen fluid was collected at 0 and 4 h after feeding. The mean intake of feed blocks and other feedstuffs offered (rice straw and concentrates) amounted to 0.3, 2.3 and 0.6 kg DM/day, respectively. Inclusion of U-CaS did not altered pH and temperature in the rumen. However, ruminal NH3-N concentration decreased quadratically (p < 0.05) in response to U-CaS inclusion, with the lowest value at 180 g U-CaS per kg feed block. With inclusion of U-CaS, the populations of rumen bacteria increased quadratically (p < 0.05) and counts of fungal zoospores were linearly enhanced (p < 0.05), being highest at 180 g U-CaS per kg feed block. Supplementation of U-CaS increased the concentration of total bacteria linearly (p < 0.05) and of Fibrobacter succinogenes quadratically (p < 0.05), whereas Ruminococcus flavefaciens and Ruminococcus albus were not affected by dietary treatments. Microbial crude protein yield and efficiency of microbial nitrogen (N) synthesis were linearly increased with different levels of U-CaS addition. Furthermore, current data clearly indicate that inclusion of U-CaS in feed blocks can affect micro-organism diversity and major cellulolytic bacteria.