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The present study exploits the potential of spent coffee grounds (SCGs) as a supplementary substrate for cultivating Pleurotus species. Increased levels of SCG in mixed substrate resulted in retardation of mycelial growth rate in a concentration-dependent manner. Subsequently, fruitification was inhibited when concentrations of SCG in the mixed substrate exceeded 30% (w/w) due to the relatively high phenolic and caffeine concentrations in SCG. Partial reductions in phenolic content and caffeine have been observed whenever SCG was present in the substrate; trace amounts of caffeine were also detected in fruiting bodies grown on all SCG-containing substrate mixtures. This finding suggests that Pleurotus spp. can reduce the levels of caffeine and phenolic compounds in SCG via bioaccumulation and biodegradation, respectively. Next, the supplementation of up to 20% (w/w) SCG did not significantly affect the proximate compositions, mineral contents, and fatty acid profiles in basidiocarps of both Pleurotus species compared to standard formulations.