Raw milk from non‐immunized cows and raw human milk from lactating mothers were examined for specificity and antibody activity against lipopolysaccharides (LPS) of five pathogenic bacteria, i.e. Escherichia coli O111:B4, E. coli O128:B12, Salmonella enteritidis, S. typhimurium and Shigella flexneri 1A in a particle concentration fluorescence immunoassay (PCFIA). Bacterial LPS was covalently coated on submicron polystyrene particles and used in an antibody sandwich technique with commercially available rabbit anti‐bovine fluorescein‐labeled IgG or goat anti‐human fluorescein‐labeled IgA. Comparison was made to a skim milk sample obtained from vaccinated cows. In general, specific activity of milk IgG against bacterial LPS did not parallel the trend of total non‐specific activity. Although the specific anti‐LPS activities were in general high in milk from vaccinated cows, milk from non‐immunized cows also showed significant activity against the bacterial LPS, sometimes higher than the vaccinated cows. IgA in human milk samples showed a wider range of antibody activity against the LPS fractions than bovine milk samples. The specificity of antibody from milk obtained from non‐immunized cows against pathogenic bacteria demonstrated that immunization of cows in order to obtain milk with high titer antibody may not be necessary as milk from non‐immunized cows can provide IgG with sufficient antibody activity against the LPS of bacterial pathogens. This can translate into significant savings and reduce stress imposed on animals for immunization.