The sea urchin Paracentrotus lividus (P. lividus) was exposed to either virgin or biofilm-covered polystyrene microbeads (micro-PS, 45 μm) in order to test the effect of microbial colonization on the uptake, biodistribution, and immune response. The biofilm was dominated by bacteria, as detected by scanning electron microscopy and 16S rRNA sequencing. A higher internalization rate of colonized micro-PS inside sea urchins compared to virgin ones was detected, suggesting a role of the plastisphere in the interaction. Colonized and virgin micro-PS showed the same biodistribution pattern by accumulating mainly in the digestive system with higher levels and faster egestion rates for the colonized. However, a significant increase of catalase and total antioxidant activity was observed only in the digestive system of colonized micro-PS-exposed individuals. Colonized micro-PS also induced a significant decrease in the number of coelomocytes with a significant increase in vibratile cells, compared to control and virgin micro-PS-exposed animals. Moreover, a general time-dependent increase in the red/white amoebocytes ratio and reactive oxygen species and a decrease in nitrogen ones were observed upon exposure to both colonized and virgin micro-PS. Overall, micro-PS colonization clearly affected the uptake and toxicological responses of the Mediterranean sea urchin P. lividus in comparison to virgin micro-PS.