Description: Plastic debris is becoming pervasive in aquatic environments, posing risks to ecosystems and human health. Environmental bacteria and fungi have shown the ability to break down plastics, influencing the transport and fate of plastic in the environment; yet few such studies mimic realistic conditions, limiting our understanding of the rates and mechanisms of these processes. While many studies have demonstrated plastics-degrading bacteria and fungi, in nature these microbes do not exist in isolation. Additionally, many studies examine the growth of microbes on plastic as their sole carbon source; however, this does not reflect environmental conditions where various carbon sources are present. Furthermore, aquatic systems are dynamic with light, temperatures, and microbial communities varying by depth, likely leading to depth-dependent microbial metabolisms and thus plastic fate. To more accurately characterize freshwater plastic degradation, our team incubated plastics at varying lake depths and maintained the resulting complex microbial biofilms in the laboratory under similar physical and nutrient conditions. We measured how microbial communities under varied conditions mobilize plastic-derived carbon (PE-C) into the environment and assimilate it into their cells using gas chromatography isotopic ratio mass spectrometry (GC-IRMS) and nano secondary ion mass spectrometry (nanoSIMS) with 99% 13C high-density polyethylene film. We found that the rates and amounts of PE-C mobilized into the environment differs between the surface photic and deep aphotic zones. Adding natural non-plastic carbon sources boosted both the mobilization of PE-C into the environment (e.g., water and atmosphere) and cellular biomass. These findings suggest that depth is a key determinant for microbial plastic degradation and that naturally occurring non-plastic carbon sources may prime microbial degraders, enhancing degradation potential in the environment. A better understanding of the microbial constraints on environmental plastic degradation is essential for modeling the fate of plastics and plastic-derived carbon, identifying favorable biodegradation conditions, and assessing potential ecosystem impacts.