In recent years, chocolate has used as an attractive and successful probiotic carrier. However, strategies are needed to maintain the survivability of probiotics in chocolate at a sufficient level throughout shelf-life, with the encapsulation of probiotics found to be a promising approach. In previous studies, cocoa powder – an essential component of chocolate – has been reported to efficiently encapsulate and protect Gram-positive probiotics such as Lactobacillus and Bifidobacterium. However, Gram-negative probiotics have not been widely tested. Escherichia coli Nissle 1917 (EcN) is a Gram-negative bacterium with probiotic properties. This experiment evaluated a novel encapsulation formulation based on cocoa powder and the potential protective effect on EcN during processing and storage. EcN was encapsulated with cocoa powder by freeze-drying for 96 hrs, followed by the evaluation of viability through serial dilution and spread plating techniques. When compared to the viable numbers before encapsulation (9.6 ± 0.22 Log CFU/g), the viable cells of EcN encapsulated by cocoa powder decreased rapidly resulting 5.4 ± 0.05 Log CFU/g at the end of encapsulation process, which is lower than the recommended minimum therapeutic numbers indicating that using cocoa powder as a solo ingredient is not highly effective on the Gram-negative probiotic EcN compared to Gram positive lactobacilli and bifidobacterial. Hence, encapsulation ability of EcN using fructooligosaccharides (FOS), whey protein concentrate (WPC) and skim milk powder with cocoa powder was evaluated as protective effect of these edible materials on Gram-positive probiotics has been demonstrated in the literature. Incorporation of cocoa powder with WPC demonstrated a positive influence in improving the survivability of EcN during the encapsulation process. EcN encapsulated by skim milk powder as a solo ingredient showed the highest viability (8.1 ± 0.58 Log CFU/g), followed by the WPC group (6.7 ± 0.05 Log CFU/g), indicating a possible role of proteins in encapsulation of EcN. Further research is needed to fully elucidate this phenomenon.