Rapidly growing fields, such as microbiome science, often lack standardization of procedures across research groups. This is especially the case for microbiome investigations in the zebrafish (Danio rerio) model system, which is quickly becoming a workhorse system for understanding the exposure-microbiome-physiology axis. To guide future investigations using this model system, we defined how various experimental decisions affect the outcomes of studies on the effects of exogenous exposure on the zebrafish gut microbiome. Using a model toxicant, benzo[a]pyrene (BaP), we assessed how each of two dissection methods (gut dissection vs. whole fish), three DNA extraction kits (Qiagen Blood & Tissue, Macherey-Nagel NucleoSpin, and Qiagen PowerSoil), and inclusion of PCR replicates (single vs. pooled triplicate reactions) affected our interpretation of how exposure influences the diversity and composition of the gut microbiome, as well as our ability to identify microbiome biomarkers of exposure. We found that inclusion of PCR replicates had the smallest effect on our final interpretations, and the effects of dissection method and DNA extraction kit had significant effects in specific contexts, primarily in the cases of identifying microbial biomarkers.