Nontarget chemical analysis using high-resolution mass spectrometry has increasingly been used to discern spatial patterns and temporal trends in anthropogenic chemical abundance in natural and engineered systems. A critical experimental design consideration in such applications, especially those monitoring complex matrices over long time periods, is a choice between analyzing samples in multiple batches as they are collected, or in one batch after all samples have been processed. While datasets acquired in multiple analytical batches can include the effects of instrumental variability over time, datasets acquired in a single batch risk compound degradation during sample storage. To assess the influence of batch effects on the analysis and interpretation of nontarget data, this study examined a set of 56 samples collected from a municipal wastewater system over 7 months. Each month's samples included 6 from sites within the collection system, one combined influent, and one treated effluent sample. Samples were analyzed using liquid chromatography high-resolution mass spectrometry in positive electrospray ionization mode in multiple batches as the samples were collected and in a single batch at the conclusion of the study. Data were aligned and normalized using internal standard scaling and ComBat, an empirical Bayes method developed for estimating and removing batch effects in microarrays. As judged by multiple lines of evidence, including comparing principal variance component analysis between single and multi-batch datasets and through patterns in principal components and hierarchical clustering analyses, ComBat appeared to significantly reduce the influence of batch effects. For this reason, we recommend the use of more, small batches with an appropriate batch correction step rather than acquisition in one large batch.