The success of mushroom production is related to several factors and production stages; among these, the preparation of substrate—responsible for the nutrition of the cultivated fungus—stands out. A quality substrate must have an ideal quantity of nutrients and be free from competing microorganisms and pests. All this information must be considered when choosing the sanitary treatment method to be adopted in commercial crops, as a nutritionally rich substrate needs to be protected by a beneficial thermophilic microbiota or be managed in sterile conditions, called axenic. Other factors are also related to the sanitary treatment of the substrate, such as operation and maintenance costs, the size of the cultivation area, the type of packaging and shelves used, the level of qualification of the workforce, the distance or difficulty in accessing spawn, etc. This book chapter will cover all the points that will clarify the best substrate production method, considering the positive and negative aspects of pasteurization and sterilization.