In vitro antimold activity of annatto natural dye against Aspergillus niger, Neurospora sitophila, and Rhizopus stolonifer was evaluated with agar well diffusion and agar dilution assays. The effects of adding annatto dye (0, 0.5, 0.75, and 1%) to bread formulations were then examined. Total microbial and fungal counts, moisture, color, radical scavenging activity, and sensory analysis of bread were monitored. A. niger was the fungus most sensitive to annatto dye in vitro. Increased dye concentrations resulted in significant decreases (P , 0.05) in moisture, total microbial and fungal counts, and L*, a*, and b* values of bread. The highest antioxidant activity was achieved in the bread with 1% of annatto dye. All bread containing annatto dye had acceptable sensory attributes as determined by panelists. The addition of annatto dye resulted in bread with a longer shelf life and acceptable sensory qualities. [ABSTRACT FROM AUTHOR]