The purpose of this study was to assess the typical microbiological quality of the most famous Portuguese traditional cheese, Serra da Estrela, and to assess its ripening time and geographical dependence. Ninety-six experimental cheeses manufactured from sixteen batches of milk on eight dairy farms scattered over the Appellation d' Origine Protégée (AOP) region were qualitatively and quantitatively evaluated microbiologically at various ripening times. Viable counts were performed after inoculation on appropriate selective media for aerobic mesophiles and proteolytic and lipolytic microflora, as well as lactococci, lactobacilli, species of Enterobacteriaceae, lactic streptococci, staphylococci, and yeasts. Members of the Enterobacteriaceae and lactic acid bacteria were the predominant microbial groups on all dairy farms throughout maturation; the latter are probably the microbial group responsible for most proteolytic and lipolytic breakdown in Serra da Estrela cheese. The microbial groups whose numbers were most affected by dairy-to-dairy variation were species of Enterobacteriaceae staphylococci, and enterococci, which are the most critical groups in terms of health hazards. It is therefore suggested that tighter control should be implemented at the level of choice of raw materials, in milk-handling practices, and in general throughout the manufacturing process in attempts to standardize production and consistently reduce microbiological risks (even though the distinctiveness of a few final organoleptic characteristics may somehow be reduced.