Abstract Aims The aim of this study was to evaluate the probiotic potential and anti-biofilm activity of five lactobacilli strains which isolated and identified from an Iranian product. Methods and Results Five lactobacilli strains, which were isolated from Zabuli yellow kashk, were evaluated for the presence of probiotic properties, such as resistance to low pH, resistance to simulated gastrointestinal conditions, bile salt tolerance, hydrophobicity, and auto- and co-aggregation. In addition, antimicrobial susceptibility, adherence to Caco-2 cells (human colon cancer cell line), anti-adhesion activity, ability against biofilm formation and biofilm degradation of mentioned strains against Pseudomonas aeruginosa PTCC 1707 were assessed. All the strains tested showed acceptable characteristics, but Lactiplantibacillus plantarum TW57-4 appeared of particular interest. Some probiotic properties of this strain were similar and in some cases higher than the commercial probiotic strain Lacticaseibacillus rhamnosus GG (standard sample). Cholesterol assimilation and radical-scavenging activity of Lpb. plantarum TW57-4 were 70.2% and 62.3%, respectively. The adhesion degree of Lpb. plantarum TW57-4 was 10.6%. Applying competition and inhibition assay, this strain showed 55.3% and 62.3% of competition and inhibition activity in adhesion of P. aeruginosa PTCC 1707 to the intestinal cells, respectively. Conclusions According to the obtained results, it can be concluded that Lpb. plantarum TW57-4 strain can be used as a promising candidate for in-vivo studies with the aim of developing new probiotic starter cultures. Significance and Impact of the Study The present study furthers our understanding of lactobacilli strains behaviour after consumption to establish their beneficial effects.