2019, National Institute of Science Communication and Information Resources (NISCAIR). All rights reserved. Cotton yarns have been pretreated with the additives, such as chitosan, microcrystalline chitosan, quaternized chitosan & aqueous extract from the fruit of Diospyros mollis Griff, as well as with the commercial formaldehyde-free cationic fixing agent (Sera® Fast C-NC) & alum (post-mordanting), and their dyeing fastness properties are studied. These treated cotton yarns are then dyed with the annatto dye extract from Bixa orellana L. (Achiote) seeds and tested for different properties including K/S value, light fastness, and wash fastness. Pre-treatment of cotton yarn with chitosan or microcrystalline chitosan solution (together with glyoxal cross-linking) or quaternized chitosan, or Sera® Fast C-NC before dyeing, shows a better color depth (K/S) and improved wash fastness properties in comparison to yarn with alum post-mordanting and the untreated cotton yarn. Improved light fastness is also obtained on inclusion of the anti-oxidant ascorbic acid in the post-treatment protocol. These additive treatments thus offer considerable potential for the improved annatto dyeing of cotton.