Serra da Estrela is the oldest and most recognizable traditional protected designation of origin (PDO) cheese from Portugal. It has been extensively studied over the years, but the latest microbial characterization is 20 years old. Hence, this work aimed to perform an updated characterization of Serra da Estrela PDO cheeses and raw materials. Our analysis showed that lactic acid bacteria content on Serra da Estrela cheeses exceeded 8.8 log CFUsg−1, in all analyzed samples, with lactococci, lactobacilli andLeuconostocspp. predominating over enterococci strains. Moreover, lactococci and lactobacilli abundance increased across the production season, while enterococci dropped considerably in late manufactures. Lastly,Leuconostocspp. content remained unchanged in all analyzed periods. A correspondence analysis showed thatL. paracasei,L. lactis,E. durans,E. faeciumandL. mesenteroidesare transversal in Serra da Estrela cheese manufacturing and were closely associated with milk, curd and cheese matrices. Additionally,L. casei,L. plantarumandL. curvatuswere specifically associated with cheese matrices, possibly active during ripening and contributing for the development of these cheeses’ organoleptic characteristics.