The formation of bacterial biofilm in dairy plant is the main source of milk and related dairy products contamination which is commonly related to improper Cleaning and ­Disinfection (C&D). Cleaning-In-Place (CIP) is the conventional process described to clean and remove on milk residues and bacterial contaminants from dairy processing lines. In this study, effectiveness of 3, 5 and 7 steps CIP protocols was evaluated against Enterococcus faecalis (E. faecalis) mature biofilms formed on stainless steel (SS) coupons. The results revealed that the most effective treatment was the 7 step protocol based on 2% sodium hydroxide (NaOH) cleaning combined with 3% enzyme mixture at 50°C for 20 minutes followed by 1% nitric acid (HNO₃) at 55°C for 20 minutes and completed by 0.5% quaternary ammonium at 25°C for 20 minutes showed a significant difference in the number of recovered cells between untreated and treated coupons with a value of 5.32 log cfu/cm² which was a very satisfying reduction level followed by CIP 2 with 4 log reduction value and CIP 1 presented by 3.14 log reduction. Even with 5 log reduction, a complete E. faecalis biofilm removal was not reached, showing persistence of mature biofilm to C&D protocols.