Between August 20, 2001, and September 17, 2002, 1429 samples including drag swabs, egg belt or egg rollout swabs, fan-blade swabs, rodent organ and intestinal pools, beetle (Alphitobius diaperinus) pools, housefly (Musca domestica) pools, chicken organ and intestinal pools, and egg pools were obtained for Salmonella culture from two flocks from two different commercial layer ranches. The two ranches were purposefully selected for the study based on their previous status of Salmonella Enteritidis isolation using environmental drag swabs in cooperation with practicing veterinarians. Salmonella sp. was isolated from 337 out of 979 (34.42%) non-egg samples. No Salmonella was isolated from 450 egg pools collected from either ranch. S. enteritidis was isolated from samples obtained from ranch 1 from manure drag swabs, 4/284 (1.4%); rodent organs, 1/24 (4.2%); and housefly pool cultures 1/21 (4.8%). Salmonella Enteritidis was isolated from ranch 2 from mouse organ and intestinal pool samples, 1/24 (4.2%). Salmonella group B was isolated from all sample types except the insects. There was a statistically significant difference in isolation rates among seven serogroups of Salmonella: groups B, C1, C2, D, E, K, and untypeable (Pearson chi-square 18.96, P = 0.002). Overall, statistically significant differences were observed with respect to Salmonella isolation among the types of samples taken (Pearson chi-square 118.54, P < 0.0001). Intensive monitoring for Salmonella Enteritidis can be used to optimize a Salmonella reduction program for an individual poultry biosecurity unit.