Listeria monocytogenes is a facultative intracellular pathogen distributed in the environment. Reference isolation methods include an enrichment step. We compared 2 protocols: one based on enrichment and the other on direct plating, with respect to the efficiency of isolation of L. monocytogenes from surface water samples. From March to May 2006, 126 surface water samples were collected biweekly from 24 discrete sampling sites located in the South Nation River basin (Ontario). Presumptive Listeria spp. were isolated using the 2 methods, and L. monocytogenes isolates were confirmed based on colony morphology and amplification of 3 pathogenicity genes: iap, inlA, and hlyA. Listeria monocytogenes was detected in 31 water samples using the enrichment approach and in 27 using the direct-plating approach, with only 13 samples positive using both approaches. No correlation was observed between the ability of the enrichment procedure to fail in the detection of the pathogenic bacterium and the abundance of several indicator bacteria. The proportion of samples carrying isolates from serovar groups 1/2a, 3a; 1/2b, 3b, 7; 4b, 4d, 4e; and Listeria spp. was significantly different between the 2 approaches. This data indicate that a direct-plating approach would be suitable for the detection of L. monocytogenes in water samples, but that enrichment and direct-plating protocols both have specific biases.