Hypercholesterolemia is a major cause of many cardiovascular and other degenerative diseases. Thus, more studies are required to discover natural sources of anticholesterol compounds. This study examined the isolation and characterization of endophytic fungi from rambusa leaves (Passiflora foetida L.) and the anticholesterol activity of endophytic fungi associated with these leaves. The endophytic fungi were separated and grown on dextrose potato agar and dextrose potato broth. The fungi were extracted with ethyl acetate and then tested with phytochemical analysis and gas chromatography-mass spectrometry (GC-MS) analysis. Fungal extracts were then studied, and anticholesterol tests were carried out using a modified Zak method. This study isolated four endophytic fungal isolates, with two fungal isolates,Colletotrichum sp., andBotrytis sp., having higher extract yields, and were selected for further testing. Further phytochemical analysis confirmed the presence of flavonoids, alkaloids, and tannins. GCMS analysis also showed the content of several fatty acid derivatives, esters, and other compounds containing amino and hydroxyl functional groups. In vitro anticholesterol test results showed that isolate Botrytis sp. had a greater effect on cholesterol reduction (24.46-39.50%) compared to Colletotrichum sp (14.16-25.17%). The results obtained provide insight into the potential of the endophytic fungiBotrytis sp. as an anticholesterol agent.