The aim of the present research was to determine the influence of sulfur and urea combined with fresh cassava root in fermented total mixed ration (FTMR) on digestibility, fermentation in the rumen, blood metabolite, milk yield, and milk quality in tropical lactating dairy cows. Four mid-lactation Thai Holstein–Friesian crossbred cows were studied. Pre-experiment milk yield was 12.7 ± 0.30 kg/day, and the body weight was 495 ± 40.0 kg. Animals were evaluated in a 2 × 2 factorial in a 4 × 4 Latin square design to receive diets followed by: factor A, which was a dose of sulfur inclusion at 1.0% and 2.0%, and factor B, which was level of urea inclusion at 1.25% and 2.5% DM in FTMR. The hydrogen cyanide (HCN) concentrations reduced 99.3% to 99.4% compared with fresh cassava root when FTMR was supplemented with 1.0% and 2.0% sulfur, respectively. Intake of crude protein was increased based on urea level addition (p < 0.05). Blood thiocyanate concentration was increased by 21.6% when sulfur was supplemented at 2.0% compared to 1.0% (p < 0.05). There was no difference in protozoal concentration, whereas bacterial populations at 4 h after feeding were significantly greater by 6.1% with the FTMR supplemented with 2.0% sulfur and 2.5% urea (p < 0.01). Allantoin concentrations, excretion, absorption, and microbial crude protein showed significant interactions between sulfur levels and urea levels in cows fed diets supplemented with 2.0% sulfur and 2.5% urea (p < 0.05). The molar ratios of the volatile fatty acid (VFA) profile were affected by dietary FTMR (p < 0.01). Furthermore, propionic acid increased by 4.6% when diets were supplemented by 2.5% sulfur (p < 0.01). Milk fat and total solids increased when feed was supplemented with 2.0% sulfur and 2.5% urea (p < 0.05). The diets supplemented with 2.0% sulfur levels resulted in greater concentrations of milk thiocyanate (p < 0.05). The somatic cell count was significantly reduced throughout the experiment with increasing sulfur supplementation (p < 0.05). Animals fed diets supplemented with 2.0% sulfur exhibited a decreased somatic cell count by 18.3% compared with those fed diets supplemented with 1.0% sulfur. Thus, inclusion of 2.0% sulfur with 2.5% urea in FTMR containing fresh cassava root improved digestibility, ruminal fermentation, microbial crude protein synthesis, and milk qualities in dairy cows.