Pleurotus mushrooms are among the most cultivated fungi in the world and are highly valuable for food, medicine, and biotechnology industries. Furthermore, Pleurotus species are carnivorous fungi; they can rapidly paralyze and kill nematodes when nutrient-deprived. The predator–prey interactions between Pleurotus and nematodes are still widely unexplored. Moreover, the molecular mechanisms and the genes involved in the carnivorous behavior of Pleurotus mushrooms remain a mystery. We are attempting to understand the interactions between Pleurotus mushrooms and their nematode prey through genetic and genomic analyses. Two single spores (ss2 and ss5) isolated from a fruiting body of Pleurotus pulmonarius exhibited significant differences in growth and toxicity against nematodes. Thus, using PacBio long reads, we assembled and annotated two high-quality genomes for these two isolates of P. pulmonarius. Each of these assemblies contains 23 scaffolds, including 6 (ss2) and 8 (ss5) telomere-to-telomere scaffolds, and they are among the most complete assembled genomes of the Pleurotus species. Comparative analyses identified the genomic differences between the two P. pulmonarius strains. In sum, this work provides a genomic resource that will be invaluable for better understanding the Italian oyster mushroom P. pulmonarius.