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The microbial basis of acidification process during spontaneous cheese whey wastewater fermentation was decrypted by implementing both culture-dependent and culture-independent techniques. Lac tobacillus and Bifidobacterium were the predominant taxa among the microbiota growing on MRS (deMan, Rogosa, and Sharpe), while Kazachstania unispora and Dekkera anomala yeast species were also isolated. Almost all Lactobacillus isolates were heterofermentative that could ferment glucose and lactose, with most of them being related to Lactobacillus hilgardii (99.0–100 % similarity). By employing fluorescence techniques, the dominance of long crescent-shaped bacteria in the acidogenic sludge was observed. Temperature gradient gel electrophoresis (TGGE), clone library, and next-generation sequencing techniques revealed the dominance of Selenomonas lacticifex. Based on Illumina data, Selenomonas in the continuous stirred-tank reactor (CSTR) represented 70.13 ± 4.64 % of the bacterial reads, while other Veillonellaceae taxa (Megasphaera and Pectinatus) represented a notable proportion (6.54 %). Prevotella was only detected by Illumina sequencing as an important constituent of the microbial population (14.97 ± 1.71 %). Budding yeasts represented 97 % of the fungal population in the CSTR, with Yarrowia strains representing 88.85 ± 5.52 % of the fungal reads. Spontaneous cheese whey acidification can favor the dominance of rumen bacteria and here was driven by the rarely reported S. lacticifex-type fermentation, which should be taken into consideration during evaluation of acidogenesis in process simulation and modelling. Moreover, the important nervonic acid content detected indicates that acidogenic sludge can be used as a source for the production of high value-added biomedical substrates.