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Antimicrobial resistance (AMR) has become one of the most serious threats to One Health. The aquatic environments are an ideal non-clinical AMR reservoir and can act as a key battlefront for tackling the AMR. However, AMR data using the One Health approach remains scarce in aquatic environments worldwide. Here, we extensively assessed AMR in E. coli isolated from urban and rural lake ecosystems using one health approach. A total of 162 E. coli isolates obtained from lakes were tested against 25 antimicrobials using an in-vitro antimicrobial susceptibility testing method. A low (2%) to moderate (45%) incidence of resistance was found for all antimicrobials used in human/veterinary medicine or in animal/plant agriculture. However,< 80% E. coli isolates exhibited MDR phenotype to highly important (amikacin, gentamicin, trimethoprim) or critically important (amoxicillin, ampicillin, colistin) drugs of both human and veterinary medicine. Of concern,> 50% of E. coli isolates exhibited multidrug resistance to drugs used as last-resorts (chloramphenicol, colistin) or as frontline (nitrofurantoin, sulfamethoxazole, ampicillin, gentamicin) against E. coli infections. In conclusion, the presence of MDR E. coli strains in urban or rural lake ecosystems highlights their possible role as AMR reservoirs or spreaders with potential One-Health risks.