Background: The endophytic fungi are an excellent source of secondary metabolites as a natural product with antimicrobial, insecticidal, cytotoxic, antioxidant, and anticancer activities. No studies on the chemical compounds of the endophytes of Mikania laevigata have been described in the literature. Objective: The objective of the study is to investigate by reversed phase high-performance liquid chromatography coupled with mass spectrometric multistage (RP-HPLC-MS/MS) the secondary metabolites of the endophytic fungi Nodulisporium sp., isolated from the medicinal plant M. laevigata (Asteraceae). Materials and Methods: The mycelia biomass strains of Nodulisporium sp. were accumulated and separated from the aqueous medium by filtration; the filtrate was subjected to a liquid–liquid partition with ethyl acetate (EtOAc) resulting in the crude EtOAc extract. This extract was analyzed by RP-HPLC-MS/MS. Results: The total mass chromatogram of the EtOAc crude strain Nodulisporium sp. extract showed compounds eluted between 13 and 20 min. The peak 1 (Rt 13.8 min), peak 2 (Rt. 14.8 min), peak 3 (Rt. 15.7 min) and peak 4 (Rt. 19.6 min) were characterized as dechlorogriseofulvin (C17H18O6), griseofulvin (C17H17O6Cl), cytochalasin D (C30H37NO6) and nodulisporic acid B2 (C43H57NO7), respectively. Conclusion: The present work provides the first scientific report on constituents of endophytic fungi Nodulisporium sp. isolated from the M. laevigata (Asteraceae).