The efficiency of Petrifilm™ AC (3M Microbiology, St. Paul, MN, USA) associated with the broths M17 and de Mann-Rogosa-Sharpe (MRS) at pH 5·4 was evaluated to enumerate Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus during the yogurt production. Commercial and reference strains of these microorganisms were experimentally inoculated in nonfat milk and incubated at 42°C for 4 h for yogurt production. At the moment of inoculation and after incubation, aliquots were collected, submitted to dilution using the broths M17 and MRS at pH 5·4, and plated for Strep. salivarius and Lb. bulgaricus enumeration according ISO 9232 and at Petrifilm™ AC plates, respectively. M17 plates were incubated at 42°C, and MRS plates were incubated at 35°C under anaerobiosis. After 48 h, the formed colonies were enumerated and the counts were compared by correlation and analysis of variance (P<0·05). In addition, colonies were randomly selected from all plates and characterized according to Gram staining and morphology. The obtained results indicated that Petrifilm™ AC plates associated to M17 and MRS at pH 5·4 can be considered as a suitable alternative for Strep. salivarius and Lb. bulgaricus enumeration during yogurt production, with slight interferences due to the acidity of MRS at the moment of inoculation, and due to the acidity of yogurt at the end of fermentation process. It was also observed that the MRS at pH 5·4 was not sufficiently selective for Lb. delbrueckii enumeration, despite it is indicated by the official protocol from ISO 9232.