Pleurotus eryngii is an edible mushroom that suffers significant losses due to fungal contamination and bacteriosis. The Pseudomonadaceae family represents one of the most frequent etiologic agents. Grapefruit seed extract (GSE) is a plant extract that contains different bioactive components, such as naringin, and exhibits a strong antibacterial and antioxidant activity. Over the last decade, GSE use as an alternative to chemical treatments in the food sector has been tested. However, to our knowledge, its application on mushroom crops has never been investigated. This study focuses on evaluating GSE efficacy in preventing P. eryngii yellowing. GSE antibiotic activity, inhibitory and bactericidal concentrations, and antibiofilm activity against several microorganisms were tested with the Kirby–Bauer disk diffusion assay, the broth microdilution susceptibility test, and the Crystal violet assay, respectively. In vitro, the extract exhibited antimicrobial and antibiofilm activity against Staphylococcus aureus 6538 and MRSA (wild type), Escherichia coli ATCC 8739, and Pseudomonas spp. (Pseudomonas aeruginosa 9027, P. fluorescens (wild type)). GSE application in vivo, in pre- and post-sprouting stages, effectively prevented bacterial infections and subsequent degradation in the mushroom crops: none of the P. eryngii treated manifested bacteriosis. Our findings support the use of GSE as an eco-friendly and sustainable alternative to chemical treatments for protecting P. eryngii crops from bacterial contamination, consequently ensuring food safety and preventing financial losses due to spoilage. Furthermore, GSE’s potential health benefits due to its content in naringin and other bioactive components present new possibilities for its use as a nutraceutical in food fortification and supplementation.