The efficiency of the disinfectants used in the milking management is fundamental to the success in the dairy activity, being a critical point to the control of mastitis. The objective was to evaluate the in vitro efficacy of iodine used in pre and post-dipping against coagulase negative Staphylococcus (CNS). Thus, 53 CNS isolates were studied for the action of the 1.0% disinfectant and their serial dilutions of 0.5%, 0.375% and 0.25%, in addition to two commercial presentations of iodine in concentrations of 0.5% and 0.25%. The rate of CNS inhibition achieved by iodine at 0.375%, 0.5% and 1.0% for 60 seconds, was 60.4%. In 30 seconds, iodine at 0.5% and 1.0% showed a microbial inhibition rate of 52.8% and 56.6%, respectively. The other protocols tested were less efficient. It is concluded that the greatest in vitro disinfectant activity for CNS was demonstrated by iodine at 0.375%, 0.5% and 1.0%, for 60 seconds. Microbial susceptibility tests should be carried out periodically, as well as health education practices and corrective training on the property, in order to ensure udder health and mastitis control.