Pichia guilliermondii can produce volatile phenols in the initial stages of wine fermentation; however, its response to different wine stress conditions has been poorly studied. In this work, we present an analysis of the response of 23 P. guilliermondii indigenous isolates to physical and chemical wine stress factors and to indigenous wine killer yeasts. Principal coordinates analysis (PCoA), based on data obtained from response patterns, was carried out to evaluate the relationships among the isolates. Major differences among the isolates were detected in media plates containing 8% ethanol and in those containing 280 g/L glucose. PCoA identified 3 clusters of isolates with different stress response patterns, indicating a relationship between the tolerance to these compounds and the origin of the isolates. Pichia guilliermondii isolates were sensitive to the toxins produced by the species Hanseniaspora uvarum, Metschnikowia pulcherrima, Wickerhamomyces anomala (ex Pichia anomala), and Pichia kluyveri, with a maximum level of sensitivity against W. anomala (91% on average). Those isolates obtained from fermenting must proved to be more resistant to killer yeasts than those obtained from grape surfaces. The combined evaluation of the response to physico-chemical and biological factors presented in this work could be a useful standard protocol for the evaluation of the potential spoilage capacity of yeasts in winemaking.