Supplementation of mushroom substrates has been linked to a higher resistance against insect pests, although few studies show the impact of this supplementation on the different agronomical parameters of mushrooms or even their chemical composition. In this work, the variation in the biological and chemical composition of oyster mushroom (Pleurotus ostreatus var. florida (Jacq.) P. Kumm) was analysed after varying the substrate supplementation of calcium silicate (0, 0.5, 1, 2, and 4% (w/w%)) during two harvest flushes. Overall, supplementation did not change the weight, the number of fruiting bodies, biological efficiency, yield ratio, and productivity rate of the mushrooms, although the harvest flushes did show significant differences. Furthermore, slight changes were found in the chemical composition with an increase in vitamin D2 and tocopherols for the mushrooms with higher amounts of calcium silicate. Overall, the substrate supplementation did not seem to induce expressive changes or decrease production yields, and can, therefore, continue to be researched as a potential application to fight agronomical pests.