When using chitosan as an antifungal agent in wood it is important to understand which factors contribute to a higher fixation ratio to optimize the utilization of chitosan, the active component. Small pine samples were impregnated with chitosan solutions varying in molecular weight, concentration, pH, polymerization agent, acid and degree of deacetylation. Different post-treatments such as time, temperature, moisture content and the effect of present air were applied to the samples to evaluate the effect on the relative retention. After impregnation, the samples, with a volume of 1.5 cm3, were leached in separate test-tubes according to EN-84. The samples were prepared in a paired design where both samples were impregnated, but only one was leached. Both leached and unleached samples were analysed for their chitosan content, and the relative ratio was used as a measure for the relative retention of chitosan during leaching. The results from these trials show that pH in the range of 5.1–5.9 is favourable. The molecular weight should be as high as possible yet able to penetrate the wood structure, and the use of acetic acid gives far better fixation than the use of hydrochloric acid.