Microplastics (MPs) are an uncontrolled contaminant affecting marine ecosystems. Studying their undesirable effects has been an attractive field for scientists in recent years. This study is the first to investigate the uptake and distribution of small microplastics (<= 3 mu m) from several sites in the Southern Mediterranean coasts. This work primarilyaims to provide a qualitative and quantitative analysis of microplastics in sediments as well as in the seaworms (Hediste diversicolor) from eight sites from the Tunisian coasts using Fourier transform infrared spectroscopy and Raman microspectroscopy. The second aim is to evaluate the potential toxic effects of environmental microplastics using a set of biomarkers such as Catalase, Glutathione-S-Transferase, Malondialdehyde and Acetylcholinesterase. Our findings showed that microplastics (1 mm-1.2 µm) were present in all sediments with its abundance ranging from 129 to 606 items /kg. Microplastic accumulation in seaworms (3 µm-0.22 µm) was 0.5-3.7 items/g. The predominant polymer was polyethylene. Results also revealed a significant variation among sites in the parameters associated with oxidative stress. Thus, size abundance of microplastics in seaworms was mainly correlated with oxidative stress biomarkers. Our data should be carefully considered in view of the microplastic presence with several types and sizes in Tunisian coastal sites, their potential toxic effects, and their transfer into food web.