The Streptococcus bovis/Streptococcus equinus complex (SBSEC) comprises pathogenic species associated with different degrees with human infections but also spontaneously fermented dairy products. We aimed therefore at developing a specific identification assay for the SBSEC targeting the 16S rRNA gene comprising a multiplex PCR followed by a differentiating restriction fragment length polymorphisms (RFLP). The multiplex PCR assay was positively applied on 200 SBSEC isolates including reference strains. The assay did not yield false-positive amplifications with strains of closely related bacteria and isolates of non-SBSEC streptococci, lactococci, enterococci, and other genera of dairy origin. The downstream RFLP using MseI and XbaI enabled further discrimination of Streptococcus infantarius/S. bovis (biotype II.1) from Streptococcus gallolyticus (biotype I and II.2)/Streptococcus alactolyticus and S. equinus. Furthermore, the newly developed primers can be used directly for Sanger sequencing. Conclusively, this novel PCR/RFLP assay is applicable in the complex dairy microbial communities and provides an important tool to assess the prevalence of members of the SBSEC in dairy products.