Listeria monocytogenes is an important foodborne pathogen with a persistent ability to form biofilm matrices in the food processing environments. In this study, we have determined the ability of bacteriophage P100 to reduce L. monocytogenes cell populations under biofilm conditions by using 21 L. monocytogenes strains representing 13 different serotypes. There were considerable differences in the ability of various strains of L. monocytogenes to form biofilms, with strains of serotype 1/2a showing maximum biofilm formation. Irrespective of the serotype, growth conditions, or biofilm levels, the phage P100 treatment significantly reduced L. monocytogenes cell populations under biofilm conditions. On the stainless steel coupon surface, there was a 3.5- to 5.4-log/cm2 reduction in L. monocytogenes cells by phage treatment. These findings illustrate that phage P100 is active against a wide range of L. monocytogenes strains in biofilm conditions.