High moisture content, low ventilation levels, and changes in the hygrothermal design of wooden dwellings could generate environmental conditions favorable for developing the rot fungus Serpula lacrymans, which is known as the most destructive fungus of its kind. The purpose of this research was to develop an experimental methodology to determine the conditions of the appearance of the fungus S. lacrymans, through in situ measurement of biological and environmental frequency indicators for wooden dwellings in South Central Chile. The referential standards for the appearance of the fungus were determined based on specialized literature and measurements of dwellings with and without deterioration. The frequency indicators for the appearance of the fungus defined and studied were as follows: frequency of air temperature FATs (%), frequency of relative humidity FRMs (%), frequency of ventilation rate FVRs (%), and rot fungus spore concentrations (CFU/pp). The methodology was validated by being measured in various enclosures and spaces under the floor in wooden dwellings in the cities of Valdivia and Temuco. The results confirmed the strong relationship between environmental relative humidity frequencies, ventilation levels, and spore concentration regarding the appearance of the fungus, where the growth dynamics cannot be separately explained without a previous analysis of the variables. In general, the dwellings most affected by the presence of the fungus had the greatest moisture levels, lower ventilation levels, and greater spore concentration. This study is the basis to diagnose the phenomena of the appearance of the rot fungus in wooden dwellings in climates favorable for its development.