The ruminant production system in Brazil is based mostly on grazing and relies on native pastures and cultivated grass pastures. Improving forage digestibility is essential to overcome the energy loss and excessive nutrient excretion by livestock. Use of exogenous fibrolytic enzymes on tropical grasses has shown promise in increasing forage utilisation and productive efficiency of ruminants. The present study was taken up to evaluate the effect of temperature and pre-incubation time of cellulose-treated substrate (CTS) and xylanase-treated substrate (XTS) on in vitro degradability of Brachiaria decumbens substrate. Two commercial fibrolytic enzymes, cellulase and xylanase, were applied at the manufacturer’s recommended dose of 7.5 and 0.46 enzymatic units per 500 mg DM, respectively. Effect of temperature regimes of substrate exposure (18°C, 25°C and 32°C) and pre-incubation times with the enzymes (0, 12 and 24 h) were investigated in a complete factorial design. Three adult rumen-cannulated Santa Inês sheep served as inoculum donors. Substrates were incubated in vitro in semi-automatic gas production (GP) system and blanks were included for each inoculum. The GP was calculated, rumen degradability and volatile fatty acids (VFA) were determined. Interaction effect between temperature of substrate exposure and pre-incubation time with the enzyme was significant on degradable neutral detergent fibre (DNDF, g/kg) for both CTS (P = 0.01) and XTS (P = 0.04). Net gas production (GP, mL/g DNDF) of XTS (P = 0.06) differ on temperature versus pre-incubation time, whereas CTS were similar (P = 0.34). Partition factor, total VFA and acetate : propionate ratio were non-significant for both the enzymes. These findings highlight that Brachiaria may respond well to exogenous application of cellulase at all the environmental conditions studied and does not need any pre-incubation period.