Isolated endophyte fungi from Mappia foetida have been explored as a potential source for the mass production of anticancer drug lead compounds in the current study. Since medical plants are not feasible economically for mass production of bioactive pharmaceutical important molecules using plant tissue culture due to factors like media design and fungal contamination, endophyte fungal mass culture have been an alternative for the relatively easy and inexpensive production. Two endophytic fungi isolated, Alternaria alternata and Fusarium species were mass cultured and their prepared alcoholic extract subjected to standard procedures to identify the phytochemical screening by gas chromatography-mass spectrometry (GCMS), high-performance liquid chromatography (HPLC), UV visible spectrophotometry (UV–VIS), and Fourier transform infrared spectroscopy (FTIR). GC–MS analysis revealed the presence of three major compounds in the extracts. The phytochemical screening confirmed the presence of an anticancer compound (camptothecin) in their extract. Moreover, the dose-dependent anticancer activity of ethanol extract was demonstrated against cervical carcinoma (HeLa), breast carcinoma (MCF-7), non-small cell lung carcinoma (H1975), and hepatocellular carcinoma cell line (Hep G2) by MTT assay where doxorubicin was used as the positive control. Furthermore, the microscopic examination also confirmed the cytotoxic effect of extract of endophytic fungi Alternaria alternata and Fusarium species against tested cancer cells. Hence, endophytic fungi Alternaria alternata and Fusarium species might be exploited for mass production of phytochemicals having anticancer activity.