A real-time (RT) optoelectronic microbiological assay for rapid detection of moulds and yeasts in yogurt was reported and validated by comparing its results with those obtained by applying the Chinese national standard method plate counting. Rapid detection of moulds and yeasts in commercialized yoghurt and spiked positive samples was successfully achieved by a semi-quantitative analytical procedure using the method, which makes it possible to pre-alert their presence or absence. The RT microbiological assay allowed detection of 10–3.5×105 CFU/mL of moulds and yeasts in 1.8–33 h. The time spent in this method for detecting microbial numbers below 10 CFU/mL as determined by conventional plate counting was over 33 h, without pre-alert during 48 h running. In addition, the results obtained closely agreed with those obtained by the plate counting method. The proposed method in this study provided a sensitive, simple and convenient approach for rapid screening of moulds and yeasts in yogurt in factories and laboratories and hence monitoring of critical control points.