Endophytic fungi are an important component of plant microbiota, and have the excellent capacity for producing a broad variety of bioactive metabolites. These bioactive metabolites not only affect the survival of the host plant, but also provide valuable lead compounds for novel drug discovery. In this study, forty-two endophytic filamentous fungi were isolated from Ficus elastica leaves, and further identified as seven individual taxa by ITS-rDNA sequencing. The antimicrobial activity of these endophytic fungi was evaluated against five pathogenic microorganisms. Two strains, Fes1711 (Penicillium funiculosum) and Fes1712 (Trichoderma harzianum), displayed broad-spectrum bioactivities. Our following study emphasizes the isolation, identification and bioactivity testing of chemical metabolites produced by T. harzianum Fes1712. Two new isocoumarin derivatives (1 and 2), together with three known compounds (3–5) were isolated, and their structures were elucidated using NMR and MS. Compounds 1 and 2 exhibited inhibitory activity against Escherichia coli. Our findings reveal that endophytic fungi from the rubber tree F. elastica leaves exhibit unique characteristics and are potential producers of novel natural bioactive products.