Postmilking teat dip is an important tool used to prevent mastitis in the modern dairy industry. In this study, we evaluated the in vitro and in vivo efficacies of a barrier teat dip containing povidone-iodine and chitosan for the prevention of mastitis. In experiment 1, we evaluated the antibacterial effects of chitosans with different molecular weights against six mastitis-causing bacteria based on the minimal inhibitory concentration test. The results showed that 50 kDa chitosan had the maximum antibacterial activity compared with 5, 150 and 350 kDa chitosans. In experiment 2, the inhibition zone test indicated that the barrier teat dip with 4.0% povidone-iodine and 1.0% chitosan had higher (P＜0.05) in vitro antibacterial efficacy against most tested mastitis-causing bacteria than the barrier teat dip with 4.0% povidone-iodine and no chitosan. In experiments 3 and 4, we evaluated the efficacies of two postmilking teat dips, 1) a barrier teat dip containing 1.0% chitosan and 4.0% povidone-iodine and 2) a conventional nonbarrier product containing 10% povidone-iodine in a field trial at two commercial dairy herds (1 and 2). A 56-d split-udder experiment (experiment 3) was conducted using 47 lactating Chinese Holstein cows in herd 1. Both left teats were immersed in barrier postmilking dip, and both right teats were dipped with nonbarrier postmilking dip. During a 56-d split-herd experiment (experiment 4), a total of 139 lactating Chinese Holstein cows from herd 2 were allocated to two groups: 1) all teats of 67 cows were dipped in the nonbarrier teat dip, and 2) all teats of 72 cows were dipped in the barrier teat dip. Milk samples were collected and analyzed for somatic cell count (SCC), fat content, protein content, and fat-to-protein ratio prior to the start of sampling (0 d), and at 28 and 56 d after initiation. Bacteriological analysis was only performed on milk samples with SCC≥200?000 cells mL-1. In experiment 3, no differences (P＞0.05) in SCC, somatic cell score (SCS) or other milk quality indicators were observed between nonbarrier and barrier teat dip treatment teats throughout the experiment. At the end of experiment 4, compared with nonbarrier teat dip group, a reduction (P＜0.05) of 29% was observed for subclinical mastitis infection prevalence in the barrier teat dip group. In the barrier teat dip group, the subclinical mastitis infection prevalence on 56 d was lower (P＜0.05) than 0 d. No differences (P＞0.05) in milk qualities or clinical mastitis incidence were detected between groups. Bacteriological analysis demonstrated that the barrier product containing povidone-iodine and chitosan reduced the subclinical mastitis infection prevalence induced by mastitis pathogens. This effect was mainly due to the reductions in Klebsiella pneumoniae , Pseudomonas aeruginosa , and Escherichia fergusonii infections. Overall, the data indicated that a barrier teat dip containing 4% povidone-iodine and 1% chitosan was more effective than 10% povidone-iodine in preventing subclinical mastitis.