Microfibers are now ubiquitous in the environment largely due to the widespread use of natural and synthetic textiles. Many enter aquatic systems through wastewater treatment plant (WWTP) effluent, surface water runoff, and atmospheric deposition, where they persist and may be ingested by filter-feeding organisms. In addition to causing physical damage (e.g., digestive and respiratory obstructions), microfibers are often carriers of chemical pollutants that may also harm biota. This exploratory study aimed to determine whether freshwater mussel (Margaritifera margaritifera L.) microfiber content varied between two rural tributaries of the Saint John River, whether microfiber content was related to WWTP discharge points or potential diffuse microfiber sources, and whether mussel size was associated with microfiber content. Mussels were collected both upstream and downstream of five WWTP discharge points and at 11 other points along two rivers within rural watersheds of maritime Canada. Microfiber content differed significantly between the two rivers; however, no trends were observed in microfiber content in relation to WWTP discharge points on either river. Smaller mussels contained significantly more microfibers than larger mussels, despite differences in mussel size ranges between tributaries. These results reveal a potential pathway for microfibers to enter aquatic food webs and highlight important implications for the use of freshwater mussels as bioindicators of microfiber contamination.