Several reports revealed that endophytic fungi have great influence on host plants, as they promote plant growth and minimize disease severity caused by various pathogens. In this study, endophytic fungi isolated from healthy plants were identified as Aspergillus terreus, Curvularia lanata, C. hawaiiensis, Macrophomina phaseolina, Fusarium solani, Talaromyces assiutensis and T. trachyspermus were evaluated for antimicrobial activity, using dual-culture plate assay and agar disc diffusion method. They have shown significant activity against root rot pathogens, Macrophomina phaseolina, Rhizoctonia solani, Fusarium solani and F.oxysporum. Talaromyces assiutensis and T. trachyspermus were selected for further study, since other fungi are well known plant pathogens or environmental contaminants. They were applied in pots and field plot experiments using sunflower as test plant. There efficacy was also compared with endophytic Cephalosporium sp., and Chaetomium sp. Talaromyces spp., significantly suppressed root rotting fungi and improved plant biomass. They ameliorated production of plant defense biochemical markers (polyphenolic content and salicylic acid) and antioxidant potential of treated plants. GC-MS profiling of n- hexane fraction of T. trachyspermus yielded several new compounds from this source. Endophytic fungi associated with healthy plants have great potential to suppress root rotting fungi and stimulate production of plant’s defense biochemical markers.