The transport and retention of Escherichia coli and bacteriophages (PRD1, MS2 and FX174), as surrogates for human pathogenic bacteria and viruses, respectively, were studied in the sand that was amended with several types of biochar produced from various feedstocks. Batch and column studies were conducted to distinguish between the role of attachment and straining in microbe retention during transport. Batch experiments conducted at various solution chemistries showed negligible attachment of viruses and bacteria to biochar before or after chemical activation. At any given solution ionic strength, the attachment of viruses to sand was significantly higher than that of biochar, whereas bacteria showed no attachment to either sand or biochar. Consistent with batch results, biochar addition (10% w/w) to sand reduced virus retention in the column experiments, suggesting a potential negative impact of biochar application to soil on virus removal. In contrast, the retention of bacteria was enhanced in biochar-amended sand columns. However, elimination of the fine fraction (< 60 μm) of biochar particles in biochar-amended sand columns significantly reduced bacteria retention. Results from batch and column experiments suggest that land application of biochar may only play a role in microbe retention via straining, by alteration of pore size distribution, and not via attachment. Consequently, the particle size distribution of biochar and sediments is a more important factor than type of biochar in determining whether land application of biochar enhances or diminishes microbial retention.