In the dairy industry, dairy by-products such as skim milk, buttermilk and butter serum which possess different specific compositions, could contact with processing surfaces to form conditioning layers and subsequently alter bacterial attachment behavior of the surfaces. In order to simulate and elucidate this process, stainless steel coupons were conditioned with skim milk, buttermilk and butter serum solutions. Formed conditioning layers were examined under a confocal laser scanning microscope (CLSM) and the influence of surface conditioning on bacterial adherence was investigated. The results showed that different conditioning layers were formed by different dairy by-products. The layer formed by skim milk, buttermilk and butter serum was the thinnest, medium and the thickest, respectively. The treatment of stainless steel surfaces with skim milk, buttermilk and butter serum could reduce the adherence of dairy-related bacteria (Lactococcus lactis subsp. lactis NBRC 100933, Leuconostoc mesenteroides subsp. cremoris NBRC 107766 and Lactobacillus casei FIRI 108) at different levels. In the majority of cases, the adherence-reducing ability of buttermilk and butter serum was found better than skim milk. While skim milk could reduce bacterial adherence during shorter exposure time (almost of 30 min), buttermilk and butter serum could act during the longer period (up to 720 min). The result suggested that, bacterial adherence-reducing effect of buttermilk and butter serum may correlate to their substances associated with milk fat globule membrane. In order to decrease bacterial adherence, surface conditioning with skim milk, buttermilk and butter serum is recommended. Surface conditioning with skim milk is suitable for short bacterial exposure time (30 min), for a longer period of time (more than 180 min), only surface conditioning with buttermilk and with butter serum is advisable. (C) 2014 Elsevier Ltd. All rights reserved.