This study investigates the biodiversity of endophytic fungi in different parts of Catharanthus roseus (L.) G. Don. and their associated chemical constituents. The research also explores the potential of these endophytes as alternative sources for industrially and medically significant secondary metabolites, including indole alkaloids. Among the twelve endophytic fungi isolated from the leaves, roots, stems, petals, and callus of the periwinkle plant, seven are reported for the first time as endophytes associated with this species. The chemical profile of ethyl acetate extracts obtained from these fungal endophytes, analyzed using Gas Chromatography-Mass Spectrometry (GC–MS), revealed the presence of various compounds, including fatty acids, alcohols, esters, and phenolic compounds. Notably, vinblastine and ajmalicine alkaloids—commonly found in the host plant—were detected and quantified in fungal extracts isolated exclusively from the roots of the plant, using Liquid Chromatography-Mass Spectrometry (LC-MS). Rhizoctonia solani was found to accumulate vinblastine (0.222 µg.g⁻¹) and ajmalicine (0.335 µg.g⁻¹), while Fusarium solani accumulated ajmalicine (0.353 µg.g⁻¹), and Acrocalymma vagum accumulated vinblastine (0.255 µg.g⁻¹). Remarkably, this is the first report of vinblastine and ajmalicine accumulation in Acrocalymma vagum and Rhizoctonia solani. Furthermore, Alternaria alternata exhibited the highest total phenolic content (10.41 mg GAE.g⁻¹), and Fusarium solani showed the highest flavonoid content (0.407 mg QE.g⁻¹). The extract of Ectophoma multirostrata demonstrated the highest antioxidant activity. These findings suggest that endophytic fungi associated with C. roseus are promising sources of biologically active metabolites with diverse therapeutic applications.

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