Gut commensals play important roles in maintaining the homeostasis of human health. Previous studies indicated that the abundance of P. goldsteinii in animal hosts was increased by the administration of prebiotics such as polysaccharides purified from iconic oriental medicinal fungi. Subsequently, P. goldsteinii was found to exert beneficial effects on the amelioration of multiple chronic inflammation-associated diseases. Even so, during the process of the development of P. goldsteinii as a next-generation probiotic (NGP), care has to be taken when it is used as a functional food ingredient. In this study, we isolated a novel P. goldsteinii strain, RV-01, from the feces of a healthy adult and carried out comprehensive analyses of its genomic and phenotypic characteristics. Bioinformatic analysis of P. goldsteinii RV-01 revealed the absence of potential virulence genes, as well as the presence of genes and traits potentially beneficial to human health, such as the production of short-chain fatty acids, anti-inflammatory lipopolysaccharides, and zwitterionic capsular polysaccharides, as well as immune regulatory proteins. To circumvent any potential side effects, the P. goldsteinii RV-01 was autoclaved before proceeding to the nonclinical safety assessment. The autoclaved P. goldsteinii RV-01 retained its anti-inflammatory effect in human colon epithelial cells. In addition to the three genotoxicity assays, 28-day subacute and 90-day subchronic animal toxicity studies (the highest dose tested was equivalent to 8.109 × 10¹⁰P. goldsteinii RV-01 cells/kg body weight/day) were also implemented. The results of all studies were negative for toxicity. These results support the conclusion that autoclaved P. goldsteinii RV-01 is safe for use as a food ingredient.