The attachment of bacteria with subsequent development of biofilms in food processing environments is a potential source of contamination that may lead to food spoilage or transmission of diseases. The present study was carried out in a buffalo dairy farm in Alexandria governorate for studying the biofilm of milking machine. A total of 192 swabs were taken from teatcups, milk jars, milk pipelines (rubber and stainless steel) and bulk tank milk (48 of each) as well as 30 bulk tank milk samples after using farm program, and after application of Iodophores and QACs. By application of Iodophores and QACs, Total bacterial counts reduced by 29.03 & 53.37; 79.90 & 80.91; 31.87 & 62.89; 55.65 & 78.03 and 66.18 & 80.37 %, respectively, While Coliforms were suppressed by 29.03 & 53.37; 79.90 & 80.91; 31.87 & 62.89; 55.65 & 78.03 and 66.18 & 80.37 %, respectively. S.aureus forming biofilm in teatcups, milk pipelines, milk jars and tank milk was reduced by 28.22 & 23.08; 67.62 & 61.62; 93.04 & 86.83 and 87.30 & 84.24 %., respectively. The reduction percentages for Enterococci counts of teatcups, milk pipelines, milk jars and tank milk and bulk tank milk after using Iodophores and QACs were 72.45 & 94.05; 74.67 & 92.13; 37.44 & 90.81; 80.25 & 95.99 and 75.46 & 79.37%, respectively. It could be concluded that QACs is more efficient on TBC, Coliforms and Enterococci counts, while Iodophores is more efficient on S.aureus. Both Iodophores and QACs are failed to overcome the microbial biofilm of milking machine.