BackgroundQuantifying infection intensity is a common goal in parasitological studies. We have previously shown that the amount of parasite DNA in faecal samples can be a biologically meaningful measure of infection intensity, even if it does not agree well with complementary counts of transmission stages (oocysts in the case of Coccidia). Parasite DNA can be quantified at relatively high throughput using quantitative polymerase chain reaction (qPCR), but amplification needs a high specificity and does not simultaneously distinguish between parasite species. Counting of amplified sequence variants (ASVs) from high-throughput marker gene sequencing using a relatively universal primer pair has the potential to distinguish between closely related co-infecting taxa and to uncover the community diversity, thus being both more specific and more open-ended.MethodsWe here compare qPCR to the sequencing-based amplification using standard PCR and a microfluidics-based PCR to quantify the unicellular parasiteEimeriain experimentally infected mice. We use multiple amplicons to differentially quantifyEimeriaspp. in a natural house mouse population.ResultsWe show that sequencing-based quantification has high accuracy. Using a combination of phylogenetic analysis and the co-occurrence network, we distinguish threeEimeriaspecies in naturally infected mice based on multiple marker regions and genes. We investigate geographical and host-related effects onEimeriaspp. community composition and find, as expected, prevalence to be largely explained by sampling locality (farm). Controlling for this effect, the novel approach allowed us to find body condition of mice to be negatively associated withEimeriaspp. abundance.ConclusionsWe conclude that amplicon sequencing provides the underused potential for species distinction and simultaneous quantification of parasites in faecal material. The method allowed us to detect a negative effect ofEimeriainfection on the body condition of mice in the natural environment.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05800-6.