Fungal endophytes display a broad range of symbiotic interactions with their host plants. Current studies on their biology, diversity and benefits are unravelling their high relevance on plant adaptation to environmental stresses. Implementation of such properties may open new perspectives in agriculture and forestry. We aim to exploit the endophytic capacities of the fungal species Fusarium equiseti, a naturally occurring root endophyte which has shown antagonism to plant pathogens, and Pochonia chlamydosporia, a nematophagous fungus with putative endophytic behavior, for plant protection and adaptation to biotic and abiotic stress. A real-time PCR protocol for quantification of the fungal population, together with Agrobacterium-mediated genetic transformation with the GFP gene for confocal microscopy analyses, were designed and applied to assess endophytic development of both these fungal species. Although quantification of both F. equiseti and P. chlamydosporia yielded similar degrees of root colonization, microscopical observations demonstrated differences in infection and development patterns. Furthermore, we found evidences of plant response against endophyte colonization, supporting a balanced antagonism between the endophyte virulence and the plant defenses. Optimization and application of the methodologies presented herein will allow elucidation of beneficial interactions among these endophytes and their host plants.