The objective of this experiment was to investigate the effect of an enzyme extract (ENZ) from Aspergillus oryzae and Aspergillus niger on in situ neutral-detergent fiber (NDF) degradability of a total mixed ration (TMR) compared with an untreated control. ENZ was evaluated at 3 TMR DM levels: 40, 50 and 60%. The TMR had 29.4% NDF and consisted of (DM basis): 57% forage and 43% concentrate feeds. ENZ was mixed with the TMR at a rate of 0.4 g per kg DM (equivalent to 10 g/cow/d at 25 kg/d DM intake) and allowed to react at room temperature for periods of 0, 6, 12, 18, and 24 h, before the ruminal in situ incubation. Bags were incubated in duplicates in 4 ruminally-cannulated Holstein cows for 0, 3, 6, 18, 24, 48, and 72 h. Cows were fed a 57% forage/43% concentrate feeds TMR with 29.4% NDF (DM basis). Rate of NDF degradability (NDFD) was estimated using LN-transformed data and the NLIN procedure of SAS. Effective NDFD (ED) was estimated using 2%/h rate of passage and assuming 0% soluble and 100% potentially degradable NDF fractions. Data were analyzed using the MIXED procedure of SAS including the fixed effects of treatment (ENZ), TMR DM, ENZ pre-incubation time, and interactions. TMR DM and pre-incubation time data were analyzed using orthogonal polynomial contrasts. Rate of NDFD decreased linearly (P ≤ 0.04) with increasing TMR DM (3.58, 2.61, and 2.78%/h, respectively; SEM = 0.28) and with increasing ENZ pre-incubation time (from 3.32 to 1.89%/h; SEM = 0.46). Compared with the untreated control, ENZ increased (P = 0.01) the rate of TMR NDFD (2.58 vs. 3.40%/h; SEM = 0.23). ED tended to decrease linearly (P = 0.07) with increasing TMR DM content and decreased quadratically (P = 0.02) with increasing ENZ pre-incubation time. ENZ increased (P = 0.02) ED compared with the control (49.8 vs. 44.0%; SEM = 1.58). There were no ENZ × TMR DM and ENZ × pre-incubation time interactions for rate of NDFD and ED (P ≥ 0.61). In this in situ experiment, enzyme treatment increased ruminal rate and effective degradability of TMR NFD.