This study evaluated the influence of coastcross hay substrate hydration and equine fecal inoculum dilution on the parameters of fermentation and microbiology in in vitro essays. A 2 × 2 factorial block design was used. The first factor was hydration of the coastcross hay substrate 12 h before incubation or at the time of incubation, and the second factor was the dilution of fecal inoculum with a nutrient solution in a weight: weight ratio of 1:1 or 1:3. Degradation of the dry matter (DM), organic matter (OM), and neutral detergent fiber (NDF) were evaluated at 24, 48, and 72 h. Microorganisms were counted 24 h after inoculation. The ammoniacal nitrogen concentration (NH 3-N), pH, and cumulative gas production were measured up to 72 h and adjusted by the non-linear Gompertz regression model. Hydration of substrate and time of incubation increased nutrient degradation of coastcross hay, as well as the final volume of gases and the concentration of Streptococcus spp. The 1:3 dilution increased the final pH and Streptococcus spp. concentration. The hydration of substrate did not have any effect on NH 3-N, Lactobacillus spp., cellulolytic, and total anaerobic bacteria concentrations. In addition, no effect of hydration was observed on the fermentation rate and the maximum fermentation time on the model used. The fermentation profile of the grass substrate is not affected by dilution, and, therefore, horse feces can be used as a source of inoculum in in vitro fermentation trials. Hydration increases the gas volumes and the nutrient degradation of grass hay, renders the lag phase time insignificant and, therefore, can be irrelevant in terms of fermentation model settings.