Anthracnose caused by Colletotrichum gloeosporioides is considered as one of the most economic vital post-harvest diseases of papaya. The present examination was directed with the objectives of assessing the potential biocontrol agents and testing their antifungal activity against Colletotrichum gloeosporioides under in vitro and in vivo. Among the different biocontrol agents, sixteen isolates of Bacillus sp. and eight Pseudomonas sp. were isolated from the fructosphere of papaya using serial dilution method and identified based on the cultural, biochemical and molecular characters. The in vitro efficacy of different biocontrol agents was tested by using dual plate techniques and the experimental results showed that the highest mycelial growth inhibition was recorded in the Bacillus sp. (BSP1) (47.83%) as compared to control (0.00 %). The defence related enzyme activity viz., peroxidase, poly phenol oxidase and catalase were studied after the treatment application and challenge inoculated with a pathogen. Effect of different treatments on fruit quality parameters viz., total sugar, reducing sugar and non-reducing sugars were analysed. The results revealed that, in pre-harvest spray with Bacillus sp. (BSP1) (5%) + post-harvest dipping with EC formulated cinnamon oil (0.1%) recorded higher content of total sugar content (14.22%) and reducing sugar (11.23%) over control and also maintained quality of papaya fruit during 14 days experimental period. All these outcomes pointed out that Bacillus spp. has tremendous potential for advancement and development of commercial formulations to control postharvest anthracnose of papaya fruit as pre-harvest spray and thus, accomplish more greater economic yield without influencing the quality.