The emergence of carbapenem resistance due to the carbapenem-hydrolyzing enzymes (carbapenemases) in Enterobacteriaceae has led to limited therapeutic options. The increased resistance to these “last-resort” antibiotics is fueled by overuse and misuse of antibiotics in human medicine and agriculture. According to the One-Health concept, the microbiomes of humans, animals and natural environments are interconnected reservoirs of antibiotic resistance genes (ARGs) and changes in one compartment will affect the other compartments. Thus, the environmental waters exposed to the pathogens, ARGs and other contaminants of human origin can play a significant role in the spread of resistance. The study aimed to characterize carbapenemase-producing Enterobacteriaceae (CPE) and ARGs in wastewaters and associated river and lake waters in Örebro, Sweden. The study also analyzed de novo development of resistance in Klebsiella oxytoca during long-term growth in river water and the effect of temperature on the emergence of resistance. OXA-48-producing Escherichia coli (ST131) and VIM-1-producing K.oxytoca (ST172) were repeatedly detected in the wastewaters and associated river, suggesting that these isolates were persistently present in these environments. Furthermore, K. oxytoca ST172 isolated from the river was genetically similar to two isolates previously recovered from patients in a local hospital, which shows the possibility of transmission of CPE from hospital to aquatic environments. A high diversity of ARGs was detected in these environments especially in hospital wastewater where ten different carbapenemase genes were detected. These results emphasized that the effective treatment of wastewaters must be ensured to reduce or eliminate the spread of antibiotic resistance. Increased resistance to meropenem (up to 8-fold) and ceftazidime (>10-fold) was observed in K. oxytoca after exposure to both river and tap water after 600 generations and resistance emerged earlier when the bacteria was grown at the higher temperature. The exposure to contaminants and increased environmental temperature may induce similar changes in the environmental microbiome, generating novel resistant variants at accelerated rates that may pose a significant threat to human health.