The objective of this study was to evaluate antimicrobial activity of chitosan microparticles (CM) in different matrices from dairy cows. CM was prepared by cross-linking of chitosan solution to enhance antimicrobial activity. Chitosan is biocompatible, natural antimicrobial biopolymer and considered Generally Recognized As Safe in many countries. Although CM shows strong antimicrobial activity in vitro, antimicrobial activity of CM in different animal matrices is not understood. We assayed antimicrobial activity of CM in cow uterine fluids (n = 3), milk samples (n = 5) and rumen fluids (n = 4) that were collected from metritic, subclinical mastitic, and fistulated dairy cows, respectively. To evaluate antimicrobial activity in the rumen fluids, we inoculated 5 × 10⁴ cfu/mL E. coli O157:H7 into the fluids, but we used endogenous microorganisms for milk and uterine samples. Concentrations of CM ranging from 0.1% to 0.8% were tested in these matrices. Efficacy of antimicrobial activity of CM was measured by direct plating of treatments to measure the concentration of pathogens. Milk and uterine fluid samples were plated on LB agar, but the rumen fluids were plated on CT-SMAC agar after 0, 2, 4 and 6 h incubation to select E. coli O157:H7. The experiment in each fluid was conducted as a completely randomized design independently and in triplicate. Data were analyzed using the GLIMMIX procedure of SAS (SAS Institute Inc., Cary, NC). Treatment with CM effectively reduced the concentration of pathogens in all of the tested samples, even though the antimicrobial activity varied depending on matrices and CM concentrations. In milk, naturally occurring pathogens were completely eliminated within 4 h with 0.1% CM (P < 0.001). Regarding the cow uterine fluid, although the growth of naturally occurring pathogens was inhibited at 0.1% (P < 0.001), higher concentration (0.6%) of CM was required to eradicate pathogens (P < 0.001). Finally, 0.2% CM reduced E. coli O157:H7 by 2 log cfu/mL in spiked rumen samples (P < 0.001). These data demonstrate that a natural antimicrobial agent, CM, retains antimicrobial activity in different matrices from dairy cows.