Anopheles mosquitoes are vectors of the human malaria parasite, Plasmodium falciparum. The vector microbiota is a likely factor influencing parasite transmission. The prokaryotic microbiota of mosquitoes is efficiently surveyed by sequencing of hypervariable regions of the 16s ribosomal RNA (rRNA) gene. However, identification of the eukaryotic microbiota by targeting the 18s rRNA gene is challenging due to simultaneous amplification of the abundant 18s rRNA gene target in the mosquito host. Consequently, the eukaryotic microbial diversity of mosquitoes is vastly underexplored. An efficient methodology is needed to identify this component of the microbiota, expected to include relatives of Plasmodium. Here, we use defined panels of Anopheles samples from West Africa to test two experimental PCR clamp approaches to maximize the specific amplification of 18s rRNA gene hypervariable regions from eukaryotic microbes: Anneal-inhibiting blocking primers and peptide-nucleic acid (PNA) oligonucleotide blockers. Of the two, PNA blockers were the only efficient blocking strategy, allowing a reduction of mosquito 18s rRNA gene sequences by more than 80% for the V4 hypervariable region. These PNA blockers will facilitate taxonomic profiling of the eukaryotic microbiota of the A. gambiae species complex, and contribute to a better understanding of microbial influence upon immunity and pathogen infection.