Technological defects in the organoleptic characteristics of cheese (odour, colour, texture and flavour) reduce quality and consumer acceptance. A red colour defect in Cabrales cheese (a traditional, blue-veined, Spanish cheese made from raw milk) occurs infrequently but can have a notable economic impact on family-owned, artisanal cheesemaking businesses. This work reports the culture-based determination of Serratia marcescens strain R01 as the microbe involved in the appearance of red spots on the surface and nearby inner areas of such cheese. Sequencing and analysis of its genome revealed a cluster of 16 genes involved in the production of prodigiosin, a tripyrrole red pigment. HPLC analysis confirmed the presence of prodigiosin in methanol extracts of S. marcescens RO1 cultures. The same was also observed in extracts from red areas of affected cheeses. The strain showed low survival rates under acidic conditions, but was not affected by concentrations of up to 5% NaCl (the usual value for blue cheese). The optimal conditions for prodigiosin production by S. marscescens RO1 on agar plates were 32 ºC and aerobic conditions. Prodigiosin has been reported to possess antimicrobial activity, which agrees with the here-observed inhibitory effect of RO1 supernatants on different bacteria, its inhibition of Enterobacteriaceae, and the delayed development of Penicillium roqueforti during cheesemaking. The association between S. marcescens and the red colour defect was strengthened by recreating the fault in experimental cheeses inoculated with RO1. The data gathered in this study points towards the starting milk to be the origin of this bacterium in cheese. These findings should help in the development of strategies that minimize the incidence of pigmenting S. marcescens in milk, the red defect the bacterium causes in cheese, and its associated economic losses.