Purpose The purpose of this study was to isolate the surface-associated microorganisms from the dairy plant surfaces with a high probability of biofilm formation and determine the most adhesive strains in terms of surface properties and exopolysaccharide production. Methods Four hundred and ninety-five surface-associated microorganisms were isolated from potential biofilm-forming surfaces of a dairy plant. One hundred and seventy of these were isolated after cleaning/disinfection of the pasteurized milk, white cheese and butter tank, yogurt and ice cream filling unit, ice cream air pressing, and condensed milk pipe. It is noteworthy that some isolates might cause post-production contamination, food infection, and intoxication. Selected 42 isolates were identified by Gram staining, physiological and biochemical tests, and 16S rRNA gene sequencing. Then, surface properties and exopolysaccharide production of 10 selected isolates were determined. To evaluate the surface properties, microbial adhesion to hydrocarbons, static water contact angle, salt aggregation, and surface zeta potential tests were performed. Result The microbial adhesion to hydrocarbons (MATH) test exhibited the lowest standard deviations, and the most consistent results between the replicates. The highest hydrophilic characteristics and exopolysaccharide production were exhibited by Gram-negative Pseudomonas aeruginosa, followed by Gram-positive Bacillus toyonensis. Also, a significant diversity of neutral sugar was determined in their alditol acetate forms by using gas chromatography-mass spectrometry. In this context, it is believed that the determination of the EPS content of the isolates would contribute to establishing an effective cleaning/disinfection procedure for dairy plants. Conclusion This study indicated that microbial adhesion is still a common problem in the dairy industry. Because of this situation, dairy plants should be organized and constructed to be suitable for hygiene and sanitary applications.