The possibility of using the diseased citrus sinensis plant as a feedstock for the synthesis of biosurfactants by various species was investigated in this study. Four fungal species were identified, namely Penicillium glabrum, Alternaria alternata, Alternaria solani, and Rhizopus stolonifer. Preliminary screening of biosurfactants was carried out by dye binding assay. About 5.75cm ring was noted for Penicillium glabrum. In the blood hemolysis assay, about halo diameter of clear zone 3.85cm was noted for Rhizopus stolonifer. The highest emulsification (E24) and oil displacement ability were noted for Penicillium glabrum and Rhizopus stolonifer. Maximum drop collapsing ability was observed for Alternaria alternata. Maximum drop sliding was observed for Alternaria solani. In the Phenol- Sulfuric acid and foaming activity assay, all four fungal species showed good activity, which indicates the presence of biosurfactants. In the microplate assay, four fungal species showed small results and minor distortion. In the penetration assay, only three species, namely, Penicillium glabrum, Alternaria alternata, and Rhizopus stolonifer, showed positive results and were able to move through hydrophilic fluids to reach the oil layer, resulting in a color change from red to hazy white.