High-Throughput Illumina Sequencing (HTS) can be used to study metagenomes, for example, those of importance for plant health. However, protocols must be optimized according to the plant system in question, the focal microorganisms in the samples, the marker genes selected, and the number of environmental samples. We optimized the protocol for metagenomic studies of aspen leaves, originating from varied genotypes sampled across the growing season, and consequently varying in phenolic composition and in the abundance of endo- and epiphytic fungal species. We optimized the DNA extraction protocol by comparing commercial kits and evaluating five fungal ribosomal specific primers (Ps) alone, and with extended primers that allow binding to sample-specific index primers, and we then optimized the amplification with these composite Ps for 380 samples. The fungal DNA concentration in the samples varied from 561 ng/mu L to 1526 ng/mu L depending on the DNA extraction kit used. However, binding to phenolic compounds affected DNA quality as assessed by Nanodrop measurements (0.63-2.04 and 0.26-2.00 absorbance ratios for 260/280 and 260/230, respectively), and this was judged to be more important in making our choice of DNA extraction kit. We initially modified the PCR conditions after determining the concentration of DNA extract in a few subsamples and then evaluated and optimized the annealing temperature, duration, and number of cycles to obtain the required amplification and PCR product bands. For three specific Ps, the extended Ps produced dimers and unexpected amplicon fragments due to nonspecific binding. However, we found that the specific Ps that targeted the ITS2 region of fungal rDNA successfully amplified this region for every sample (with and without the extension PP) resulting in the desired PCR bands, and also allowing the addition of sample-specific index primers, findings which were successfully verified in a second PCR. The optimized protocol allowed us to successfully prepare an amplicon library in order to subject the intended 380 environmental samples to HTS.