Plant material obtained by pruning and production of deciduous fruit trees was evaluated as substrates for the production of the oyster mushroom, Pleurotus ostreatus . Lignified branches and stems from peach, apple, and pear trees were processed using a ripping machine to reduce the size of chips and to optimize disinfection. A completely randomized experimental design was proposed with six treatments (novel substrates) and one absolute control (100% hay substrate). Morphological variables such as thickness and diameter of the pileus, stipe length were assessed, as well as production variables (sprouting, fresh weight, and biological efficiency) and bromatological analysis (ash, ethereal extract, crude fiber, and crude protein). Apart from the 100%-hay substrate, biological efficiency ranged between 27% (100% apple tree as the substrate) and 140% (50% hay + 50% peach tree as the substrate). According to morphological analysis, the highest diameters were recorded from mixed substrates (50% hay + 50% wood), and a highly significant positive correlation was found between diameter and stipe length. Morphological parameters were not significantly correlated with biological efficiency. Analysis of biological efficiency confirmed that mixed substrates clustered together with 100%-wood substrates. Bromatological analysis showed that the mixed substrate (50% hay + 50% pear tree) had the highest protein content among the novel tested substrates. Bromatological parameters were not significantly correlated with biological efficiency. In conclusion, pruning residues from fruit trees can be valuable by using them as substrates for the cultivation of P. ostreatus . Production is quantitatively competitive with that using hay, on the condition that wood is mixed with hay.