AbstractBiofilm is considered as a community of microorganisms in which cells adhere to each other on surfaces in a self‐produced matrix of extracellular polymer compounds. In recent years, efforts to use the beneficial aspects of biofilm in probiotic research have intensified. In this study, probiotic biofilms ofLactiplantibacillus plantarumandLacticaseibacillus rhamnosuswere manufactured using milk and transferred to yogurt in whole and pulverized forms to test in real food conditions. Survival was assessed during 21 days of storage time as well as gastrointestinal conditions. The results indicated thatLp. plantarumandLc. rhamnosuscan form a very desirable and strong biofilm that can have a good protective effect on the survival of these bacteria in probiotic yogurt during processing, storage, and gastrointestinal conditions, in a way that, after 120 min of treatment in high acidic gastrointestinal conditions (pH 2.0), the survival rate decreased by only 0.5 and 1.1 log CFU/ml. Probiotic biofilm can be used as a natural way of utilizing bacteria in biotechnology and fermentation, which is an excellent way to increase the utility of probiotics.