The presence of plastic particles in oceans has been recognized as a major environmental concern. The decrease in particle size increases their ability to directly interact with biota, with particles in the nanometer size range (nanoplastics—NPs) displaying a higher ability to penetrate biological membranes, which increases with the decrease in particle size. This study aimed to evaluate the role of life stages in the effects of poly(methyl)methacrylate (PMMA) NPs on the polychaete Hediste diversicolor, a key species in the marine food web and nutrient cycle. Thus, behavioral (burrowing activity in clean and spiked sediment) and biochemical endpoints (neurotransmission, energy reserves, antioxidant defenses, and oxidative damage) were assessed in juvenile and adult organisms after 10 days of exposure to spiked sediment (between 0.5 and 128 mg PMMA NPs/Kg sediment). Overall, the results show that H. diversicolor is sensitive to the presence of PMMA NPs. In juveniles, exposed organisms took longer to burrow in sediment, with significant differences from the controls being observed at all tested concentrations when the test was performed with clean sediment, whereas in PMMA NP-spiked sediment, effects were only found at the concentrations 8, 32, and 128 mg PMMA NPs/Kg sediment. Adults displayed lower sensitivity, with differences to controls being found, for both sediment types, at 8, 32, and 128 mg PMMA NPs/Kg sediment. In terms of Acetylcholinesterase, used as a marker of effects on neurotransmission, juveniles and adults displayed opposite trends, with exposed juveniles displaying increased activity (suggesting apoptosis), whereas in adults, overall decreased activity was found. Energy-related parameters revealed a generally similar pattern (increase in exposed organisms) and higher sensitivity in juveniles (significant effects even at the lower concentrations). NPs also demonstrated the ability to increase antioxidant defenses (higher in juveniles), with oxidative damage only being found in terms of protein carbonylation (all tested NPs conditions) in juveniles. Overall, the data reveal the potential of PMMA NPs to affect behavior and induce toxic effects in H. diversicolor, with greater effects in juveniles.