Antilisterial bacteriocin producing strain were isolated from milk samples and were subjected to 16S rRNA sequencing and found to be of genus Enterococcus faecium. The bacteriocin ALC102 were partially purified by Amberlite XAD-16 adsorption followed by column chromatography. The biofilm formation capacity of Listeria monocytogenes MTCC 657 were evaluated by tube method and CV binding assay. Biofilm formation on different abiotic substrates were also evaluated. Among three substrates stainless steel had a strong biofilm formation followed by glass and aluminum foil. From the results of biofilm eradication studies, the bacteriocin ALC102 showed almost similar activity of commercial bacteriocin nisin on all the substrates at 45°C, 30°C, 4°C and -20°C. Based on CBD® biofilm eradication assay, the eradication potential of ALC102 and nisin were found to be similar on high (45°C) and freezing (-20°C) temperatures. From the study, antilisterial bacteriocin ALC102 found to be able to inhibit the biofilm formed Listeria monocytogenes MTCC 657 at different temperatures and different incubation periods (24h, 48h and 72h). The biofilm eradication potential of antilisterial bacteriocin ALC102 was similar to nisin. Neither incubation temperature nor incubation period doesn’t altered the activity of the bacteriocin. So this bacteriocin can be considered as a potential competitor in food industry and we strongly recommend the use of this bacteriocin from Enterococcus faecium GRD AA in the food preservation industry to a higher temperature (45°C) to freezing temperature (-20°C).