The use of red grape pomace (GP; Vitis vinifera L. var. Shiraz) as a source of beneficial bioactive compounds in ruminant diets is limited by high levels of indigestible compounds in the grape skin matrix. This problem demands innovative, inexpensive, and easy-to-use strategies that improve the digestibility of GP. The bioconversion of GP using edible oyster mushrooms (Pleurotus ostreatus) is one such strategy that has not been previously explored. Therefore, this study evaluated the effect of cultivating oyster mushrooms on GP on chemical composition and in vitro ruminal fermentation parameters of the spent mushroom substrate. The GP was inoculated with oyster mushroom spawns at 0, 200, 300, 400, or 500 g/kg, and incubated for 4 weeks. Organic matter, acid detergent lignin, sodium, manganese, cobalt, and copper linearly declined (P < 0.05) as spawn rates increased. A quadratic trend was observed for crude protein, neutral detergent fibre, acid detergent fibre, magnesium, phosphorus, and calcium content in response to increasing spawn rates. Higher spawning rates (20-50%) had a positive effect (P < 0.05) on gas production from the immediately fermentable fraction (a), rate of gas production from the slowly fermentable fraction (c) and effective gas production. However, gas production from the slowly fermentable fraction (b) and potential gas production linearly declined in response to increasing spawning rates. There was a linear increase (P < 0.05) in the immediately degradable fraction (a), while quadratic effects were observed for partition factors, effective degradability, and in vitro organic matter degradability at 48 h in response to spawning rates. It can be concluded that inoculating GP with oyster mushroom spawn reduced fibre content while increasing crude protein content and in vitro ruminal fermentation efficiency of red grape pomace. Based on the quadratic responses of partition factors at 48 hours post-inoculation, the optimum spawning rate for maximum ruminal fermentation efficiency of GP was determined to be 300 g/kg.