Toxicology research into the global public health burden of fine particulate matter (PM2.5) exposures frequently requires extraction of PM2.5 from filters. A standardized method for these extractions does not exist, leading to inaccurate interlaboratory comparisons. It is largely unknown how different filter extraction methods might impact the composition and bioactivity of the resulting samples. We characterized the variation in these metrics by using equal portions of a single PM2.5 filter, with each portion undergoing a different extraction method. Significant differences were observed between extraction methods for concentrations of elements and polycyclic aromatic hydrocarbons (PAHs) for the PM2.5 tested following its preparation for biological response studies. Importantly, the chemical profiles differed from those observed when we used standard protocols for chemical characterization of the ambient sample, demonstrating that extraction can alter both chemical component amounts and species profiles of the extracts. The impact of these chemical differences on sensitive end points of zebrafish development was investigated. Significant differences in the percent incidence and timing of mortality were associated with the PM2.5 extraction method. This research highlights the importance of and rationale for considering the extraction method when interlaboratory comparisons of PM2.5 toxicology research are made.