Changes in the microflora and biochemical characteristics of Afuega’l Pitu cheese were studied throughout a 60-d ripening period. Total viable counts were high in milk (7.62±0.95 log₁₀ cfu/ml) and in cheese throughout ripening (9.26±0.56 log₁₀ cfu/mg and in 3-d-old cheese; 8.06±0.47 log₁₀ cfu/g in 60-d-old cheese). Lactic acid bacteria were the main microbial group responsible for the substantial pH decrease during coagulation and for the further maintenance of low pH. Lactococcus lactis ssp. lactis, citrate-utilizing Lactococcus lactis ssp. lactis, Leuconostoc mesenteroides ssp. mesenteroides, Leuconostoc mesenteroides ssp. dextranicum, Leuconostoc citreum, Leuconostoc paramesenteroides, Lactobacillus plantarum, and Lactobacillus brevis were the species identified. Mean TS and the percentage of salt in the moisture phase increased significantly during ripening to reach 72.32 and 7.51%, respectively, in 60-d-old cheeses. The mean fat and protein contents were 46.67% and 41.77% of TS, respectively. Proteolysis was weak, water-soluble N attained 16% of total N at the end of ripening, and phosphotungstic acid-soluble N increased from 0.36 to 2.12% of total N during 60 d of ripening. The β-CN remained relatively intact, and, consequently, the γ-CN fraction did not significantly increase. Residual α_sl-CN also remained quite high throughout ripening compared with other varieties of traditional raw milk cheeses.