Context Feeding of fresh cassava root in ruminants is limited because it contains a high level of hydrocyanic acid (HCN), which is responsible for poisoning. Aims The objective of the present study was to evaluate the effect of sulfur levels supplementation in the fermented total mixed ration (FTMR) containing fresh cassava root as an energy source on the gas kinetics, ruminal fermentation, reduction of HCN concentration and nutrient digestibility in the in vitro gas production. Methods The experimental design was a 3 × 4 factorial in a completely randomised design. Dietary treatments contained factor A, which was three levels of sulfur supplementation at 0, 1 and 2% in FTMR, and factor B was ensiling time at 0, 7, 14 and 21 days respectively. Key results Concentration of HCN in FTMR was significantly reduced (P < 0.05) by 73.7% when sulfur was supplemented in FTMR at 2%. The levels of HCN in FTMR were 2.89, 0.61, 0.61 and 0.49 ppm, for ensiling time of 0, 7, 14 and 21 days, respectively (P < 0.01). HCN was reduced when ensiling started at 7 days. Gas production from soluble fractions (a) ranged from –1.2 to –2.4 and was not significant (P > 0.05). Furthermore, gas production from the insoluble fraction (b) ranged from 48.8 to 53.9, and gas production rate constants for the insoluble fraction (c) ranged from 0.1 to 0.2. The potential extent of gas production (a + b) was also unchanged when the concentration of sulfur increased (P > 0.05). In addition, there were no interactions between sulfur levels and ensiling times on all parameters (P > 0.05). In contrast, cumulative gas production (at 96 h of incubation) was significantly different when sulfur increased at 2% (P < 0.05), while ensiling times did not affect cumulative gas production. Ruminal pH was affected by FTMR and decreased with an ensiling time of 21 days, ranging from 6.0 to 6.1 after ensiling. Ensiling time did not affect ruminal ammonia-nitrogen concentration (P > 0.05) among dietary treatments which ranged from 21.2 to 24.0 mg%. FTMR ensiled for 21 days had the highest in in vitro dry matter digestibility, an in vitro neutral detergent fibre and in vitro acid detergent fibre digestibility which were 61.0–62.5, 35.1–43.1 and 22.3–25.9% dry matter (DM) respectively. Regarding the concentration of total volatile fatty acid (VFA), acetic acid, propionic acid and butyric acid, ranges from 94.7 to 113.6 mmol/L, 59.3 to 67.4, 20.2 to 25.9 and 11.3 to 13.8 mol/100 mol, respectively, were observed and did not differ among treatments (P > 0.05). The concentration of total VFA relative to the sulfur level and ensiling time had no effect on ruminal VFA concentrations. However, exceedingly high percentages of sulfur (2% of the DM) in the diet tend to increase total VFA concentration. Conclusions Using of 2% sulfur supplementation in TMR containing fresh cassava root fermented could improve the kinetics of gas and nutrient digestibility while maintaining ruminal fermentation parameters and the rate of HCN disappearance. Implications These findings should be examined in further in vivo experiments in order to increase animal performance.