Fungal growth and mycotoxin production is the major economic importance in bakery products during manufacturing and storage. So, the present study aimed to prepare chitosan loaded mixed nano-fermentate of three Lactobacillus sp. [L. plantarum, L. helveticus and L. rhamnosus (PHR)] to be used as bio-preservatives in pan bread and cupcake. PHR-nanofermentate were applied in bread samples during mixing the ingredient, injected in bread loaves at the end of fermentation or added as coating materials after baking. Cupcake samples were treated by PHR-nanofermentate during mixing the ingredients or as coating materials after baking. Baking quality, color parameters, sensory properties and mold count of bread and cupcake samples were performed. Bread sample coated with PHR- nanofermentate after baking ranked the highest between the treated samples. Cake samples treated with PHR- nanofermentate during mixing step had higher scores for all sensory attributes compared to the coated cake, except the texture and appearance parameters. PHR-nanofermentate resulted in more effective disappeared mold counts at the end of shelf life for bread and cake. In conclusion, PHR-nanofermentate achieved the intended galas as antimicrobial agent and prolonged the shelf-life, regardless the addition method, in chemically leavened products. However, its addition method to bread and other baked products which utilize baker's yeast as a leavening should be considered. Finally, this study demonstrates that "PHR" is a good product for the bio-preservation of food.