Pleurotus fungi, also known as basidiomycetous fungi, have been a part of human culture for thousands of years. They exhibit anticancer, antitumor, antibacterial, and immunomodulatory effects, having biotechnological, medicinal, and aesthetic applications. They are also versatile, highly resistant to illnesses and pests, and do not require special growing conditions. These properties make them readily marketable, and can be found in supermarkets worldwide, generating multimillion-dollar sale revenues. The global edible mushroom market was valued at USD 5.08 billion in 2021, which is expected to grow to USD 6.43 billion in 2028. China produces about 87% of Pleurotus spp. globally; other Asian countries generate 12%, and Europe and America account for approximately 1%. Pleurotus spp. have distinct functional characteristics, including high protein content with a proper essential amino acid score pattern, dietary fibre profile, high amounts of vitamins (e.g., B and D) and minerals (e.g., Fe, Zn, Cu, and Se), and low fat. Therefore, Pleurotus spp. can provide alternative industrial tools. The present review discusses Pleurotus spp. as biotechnological tools for acquiring metabolites of interest, studying them, and analysing bioactive substances that can be used in various fields, including medicine and food.