One of the major problems in industrial water systems is the generation of biofilm, which is resistant to antimicrobial agents and causes failure of sanitization policy. This work aimed to study the anti-biofilm activity of peracetic acid (PAA) at contact times and temperatures combinations. To this end, a 96 well microtiter-based calorimetric method was applied in in vitro biofilm production using Ecsherichia coli, isolated from the water supply system of a pharmaceutical plant. The phenotypic and phylogenetic tests confirmed the isolated bacteria belong to strains of Ecsherichia coli. The anti-biofilm activity of peracetic acid on formed biofilm was investigated at concentrations of 0.15-0.5% for a contact time of 5-15 min at 20°C to 60°C. The maximum biofilm formation by MTP method using an Ecsherichia coli isolate was achieved in 96 h incubation in TSB containing wells at 37°C. Biofilm formation rate showed to be high by the environmental isolate compared with that of standard strain. PAA at concentrations above 0.25%, the temperature of 40°C, and a minimum of 10 minutes of contact time was effective in the eradication of biofilm in an MPT-based system.