Background: Endophytic fungi that live asymptomatically inside the plant tissues have novel bioactive metabolites exhibiting a variety of biological activities, especially against cancer. Cordia dichotoma G. Forst. play a significant role in traditional medicines and therapeutics. Leaves and bark have been used as anti-inflammatory and anticancer agents. Objectives: Isolation, screening, and in silico toxicity evaluation of camptothecin (CPT) from the endophytic fungus Pestalotiopsis microspora MH458929. Materials and Methods: Endophytic fungus was isolated from leaves of C. dichotoma collected from Sathyamangalam Tiger Reserve forest (STRF), Tamil Nadu. The wild strain was identified by 18S rDNA sequencing. Modified potato dextrose broth was used as a screening medium for the presence of CPT. CPT was analyzed by high-performance liquid chromatography and electrospray ionization–mass spectrometry (ESI-MS). Compounds identified by ESI-MS from fungal extract were further studied for their in silico toxicity study against Daphnia magna, Tetrahymena pyriformis, Pimephales promelas, and Rattus sp. Bioaccumulation factors, developmental toxicity, and mutagenicity were studied by the quantitative structure–activity relationship model – Toxicity Estimation Software Tool. Results: Endophytic fungus P. microspora produced a maximum yield of 0.691 mg/L of CPT. CPT derivatives were identified at m/z of 349.10, 363.08, and 389.41 through ESI-MS analysis. In silico toxicity study revealed that compounds were of Category D and hence considered nontoxic to higher organisms. However, compounds showed high toxicity for lower organisms, with toxicity order D. magna > T. pyriformis > P. promelas > rat. Conclusion: The present study is the first report to screen, isolate, and analyze the CPT's in silico toxicity and its derivatives from endophytic fungus P. microspora from STRF. Further in vitro and in vivo studies are recommended to utilize CPT and its derivatives in pharmaceuticals.