Freshwater ecosystems are increasingly impacted by chemical stressors, with microplastic (MP) pollution emerging as a significant threat to aquatic life. Bivalves, such as mussels, are widely used in biomonitoring programs to assess ecological pressure from MPs. This study investigates the impact of MP accumulation on oxidative stress biomarkers in freshwater mussels (Unio elongatulus) from Sacchetti Ditch, a lowland spring watercourse in Northwestern Italy. Over one-year period, levels of several oxidative stress biomarkers were measured, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), glyoxalases I (GI) and glyoxalase II (GII), lactate dehydrogenase (LDH), and metallothioneins (MTs) in both the gills and digestive gland of mussels, which were sampled every two months. These biomarkers were correlated with MP bioaccumulation in the same mussels. MPs were detected in all sampled mussels, with filament sizes ranging from 110-155 µm being the most common, and white, black, blue, and red being the predominant colors. Polypropylene, polyethylene, polyethylene terephthalate, and polyamide were the dominant polymers MP concentrations remained low but stable throughout the year, with slightly higher levels observed during warmer months. The study revealed that GII and LDH in the digestive gland, as well as GST in the gills, were most affected by the MP load, indicating oxidative stress. Correlations revealed a negative relationship between MP levels and GST in the gills and LDH in the digestive gland, while a positive correlation was found with GII in the latter tissue. The response of other biomarkers, such as SOD, CAT, GPx, GR, GI, and MTs, suggests that additional environmental contaminants or ecological changes may also play a role. These findings underscore the ongoing ecological pressure on mussels in lowland spring watercourses, particularly those impacted by human activities like Sacchetti Ditch.