Abstract Plastics have been produced for over a century, but definitive evidence of complete plastic biodegradation in different habitats, particularly freshwater ecosystems, is still missing. Using 13 C‐labelled polyethylene microplastics (PE‐MP) and stable isotope analysis of produced gas and microbial membrane lipids, we determined the biodegradation rate and fate of carbon in PE‐MP in different freshwater types. The biodegradation rate in the humic‐lake waters was much higher (0.45% ± 0.21% per year) than in the clear‐lake waters (0.07% ± 0.06% per year) or the artificial freshwater medium (0.02% ± 0.02% per year). Complete biodegradation of PE‐MP was calculated to last 100–200 years in humic‐lake waters, 300–4000 years in clear‐lake waters, and 2000–20,000 years in the artificial freshwater medium. The concentration of 18:1ω7, characteristic phospholipid fatty acid in Alpha‐ and Gammaproteobacteria, was a predictor of faster biodegradation of PE. Uncultured Acetobacteraceae and Comamonadaceae among Alpha‐ and Gammaproteobacteria, respectively, were major bacteria related to the biodegradation of PE‐MP. Overall, it appears that microorganisms in humic lakes with naturally occurring refractory polymers are more adept at decomposing PE than those in other waters.