Okra (Abelmoschus esculentus) is a widely cultivated vegetable crop that is susceptible to various pathogens, including Fusarium oxysporum, which causes severe disease of wilt. Chemical control using fungicides currently poses risks to human and animal health. To find ecofriendly method, the present study aimed to compare the effectiveness of different bio-control agents (BCAs) against Fusarium wilt in Okra for eco-safe management. Okra seeds were taken from Vegetable Research Institute, Ayub Agricultural Research Institute. Disease samples were collected, and the pathogen was isolated, purified, and multiplied by using potato dextrose agar media. Pure culture was used for the isolation of DNA and amplification of the ITS region was accomplished by using ITS-1 and ITS-4 primers. The fragments were visualized by running it on 1% agarose gel. The PCR product of F. oxysporum pv. vasinfectum was successfully confirmed based on size of fragment by comparing with 1 kb marker. It was verified that the selected primer pair specifically amplified the anticipated 18S rDNA fragment, measuring 500 base pairs in length. Moreover, in-vitro tests revealed that BCAs like Bacillus spp. inhibited Fusarium fungus by 41.2%, while Trichoderma inhibited it by 38.5%. Control groups exhibited no inhibition of pathogen growth. Taken together, this effort has important implications for the implementation of biological management strategies in Okra field to mitigate the impact of Fusarium wilt disease.