The antimicrobial action of chitosan against wine related microorganisms, including Lactobacillus plantarum, Saccharomyces cerevisiae, Oeonococcus oeni, Lactobacillus hilgardii, Brettanomyces bruxellensis, Hanseniaspora uvarum and Zygosaccharomyces bailii was examined in laboratory media. In order to assess the potential applicability of chitosan as a microbial control agent for wine, the effect of chitosan, applied individually and/or in combination with sulphur dioxide (SO2), on the growth of microorganisms involved in various stages of winemaking and on the fermentative performance of S. cerevisiae was investigated. Of the seven wine-related microorganisms studied, S. cerevisiae exhibited the strongest resistance to antimicrobial action of chitosan in laboratory media with a minimum inhibitory concentration (MIC) greater than 2 g/L. L. hilgardii, O. oeni and B. bruxellensis were the most susceptible to chitosan since they were completely inactivated by chitosan at 0.2 g/L. The MIC of chitosan for L. plantarum, H. uvarum and Z. bailii was 2, 0.4 and 0.4 g/L, respectively. In wine experiments, it was found that chitosan had a retarding effect on alcoholic fermentation without significantly altering the viability and the fermentative performance of S. cerevisiae. With regard to non-Saccharomyces yeasts (H. uvarum and Z. bailii) involved in winemaking, the early deaths of these yeasts in mixed cultures with S. cerevisiae were not probably due to the antimicrobial action of chitosan but rather due to ethanol produced by the yeasts. The complex interactions between chitosan and wine ingredients as well as microbial interactions during wine fermentation considerably affect the efficacy of chitosan. It was concluded that chitosan was worthy of further investigation as an alternative or complementary preservative to SO2 in wine industry.