Many root fungal endophytes inhabiting forest trees have potential impact on the health and disease progression of certain tree species. Hence, the screening of root endophytes for their biocontrol abilities is relevant for their potential to protect their hosts against invaders. The aim of this research is to screen for the potential inhibitory effects of selected conifer root endophytes during interaction, in vitro, with the root rot pathogen, Heterobasidion parviporum. Here, we introduce a guideline that facilitates the use of root fungal endophytes as biocontrol agents. We isolated fungal root endophytes from eight different conifers. These root fungal endophytes were evaluated for their antagonism against the root rot pathogen, H. parviporum, by means of paired-culture antagonism assays. We determined the antagonism of the isolated root fungal endophytes to elucidate potential biocontrol applications. For the analysis, a software package in R was developed. Endophyte candidates with antagonistic potential were identified.