Peronema canescens Jack has shown promising anticancer activities. This study set out to investigate the rich biodiversity and potential anticancer properties of fungal endophytes derived from P. canescens. Forty-eight pure isolates of fungal endophytes were obtained from cultures of both young and old leaves across eleven different branch types. Furthermore, four specific fungal strains were selected for the evaluation of anticancer activity: Fusarium sp. strain PCL-34-B, Phomopsis sp. strain PCL-Y-48, Lasiodiplodia sp. strain PCL-O-9, and Phomopsis sp. strain PCL-O-23. The ethyl acetate and methanol extracts of these fungal endophytes demonstrated intriguing results, with the ethyl acetate extracts of PCL-Y-48 exhibiting the most significant cytotoxic activity among the various fungal endophyte extracts. The half-maximal inhibitory concentration values were recorded at 10.52 and 33.54 mg/L against the A549 lung cancer and MG-63 osteosarcoma cells, respectively. The identification of chemical substances reveals a range of components believed to play a role in their anticancer effects.