In order to discover the main microbial populations present throughout the process of beet sugar extraction and to identify the steps where possible control measures could be implemented, four Spanish beet sugar extraction factories were investigated. Eighteen stages were sampled at each factory and analyzed for several microbial groups and physicochemical parameters. Beets, cossettes, and diffusion juices were the most contaminated samples during beet sugar extraction, and mesophiles, thermophiles, and yeasts were the most numerous microbiota. Differences in contamination among factories and among stages of the sugar extraction process were statistically significant (P < 0.01). Beets and cossettes showed high numbers of mesophiles and lactic acid bacteria, with gum-forming bacteria and yeasts being at levels of >6 log CFU/g. Diffusion juices were highly contaminated with aerobic mesophiles, lactic acid bacteria, and thermophiles. Beet-washing water, equipment, and air were also important sources of contamination; in contrast, diffusion water showed moderate levels of microorganisms due to the acid treatment performed. Gum-forming bacteria, which are present at high levels from the first step of the extraction, survive the diffusion process and remain in the diffusion juice, where they may produce "gums" responsible for obstruction of filters. Thermophiles were present, although at levels lower than expected in most samples. Other microbial groups, particularly yeasts, may also contribute to the destruction of sugar. No biocides were used during the beet sugar extraction in the factories investigated.