Biofilms are the most common mode of bacterial growth in nature and the formation will occur on organic or inorganic solid surfaces in contact with a liquid. The aims of this study were, by combining numeration and sessile drop technique, (i) to characterize the structural dynamics of dairy biofilm growth and the physico chemical properties on silicone and stainless steel and (ii) to evaluate the impact of bio-adhesion on chemistry of surfaces at different times of contact (2, 7, 9 and 24h). Significantly, greater biofilm volumes were observed after 48h on two materials. Gram-positive bacteria and fungal population exhibited a significantly higher biofilm organization than gram-negative (4364%). Elsewhere, after 48h, results showed a slight difference on gram-negative adhered cells on stainless steel than silicone (2.6x107cfu/cm2 and 4.7x105cfu/cm2, respectively). Moreover, the physico chemical properties of the surfaces showed that the silicone and stainless steel have a hydrophobic character (Giwi=68.28mJ/m2 and 57.6mJ/m2, respectively). Also, both the surfaces present a weak electron donor character ( =2.2mJ/m2 and 4.1mJ/m2, respectively). The real-time investigation of the impact of dairy biofilm on the physico chemical properties of the materials has shown a decrease of hydrophobicity degree of the silicone surface that becomes hydrophilic (Giwi=11.47mJ/m2) after 7h and the increase of electron donor character ( =75.8mJ/m2). Elsewhere, bio-adhesion on stainless steel was accompanied with a decrease of hydrophobicity degree of the surface, which becomes hydrophilic after 7h of contact (Giwi=6.62mJ/m2) and the increase of the electron donor character ( =44.8mJ/m2). While, after 24h of contact, results showed a decrease of the hydrophilicity degree and surface energy components of silicone and stainless steel that become hydrophobic (Giwi=21.2mJ/m2 and Giwi=56.51mJ/m2, respectively) and weak electron donor ( =14.0 and 2.3mJ/m2, respectively).