Infections by Extended-Spectrum Beta-Lactamase producing Escherichia coli (ESBL-Ec) are on the increase in Ghana, but the level of environmental contamination with this organism, which may contribute to growing Antimicrobial Resistance (AMR), is unknown. Using the WHO OneHealth Tricycle Protocol, we investigated the contamination of E. coli (Ec) and ESBL-Ec in two rivers in Ghana (Odaw in Accra and Okurudu in Kasoa) that receive effluents from human and animal wastewater hotspots over a 12-month period. Concentrations of Ec, ESBL-Ec and percent ESBL-Ec/Ec were determined per 100 mL sample. Of 96 samples, 94 (98%) were positive for ESBL-Ec. concentrations per 100 mL (MCs100) of ESBL-Ec and %ESBL-Ec from both rivers were 4.2 × 10⁴ (IQR, 3.1 × 10³–2.3 × 10⁵) and 2.79 (IQR, 0.96–6.03), respectively. MCs100 were significantly lower in upstream waters: 1.8 × 10⁴ (IQR, 9.0 × 10³–3.9 × 10⁴) as compared to downstream waters: 1.9 × 10⁶ (IQR, 3.7 × 10⁵–5.4 × 10⁶). Both human and animal wastewater effluents contributed to the increased contamination downstream. This study revealed high levels of ESBL-Ec in rivers flowing through two cities in Ghana. There is a need to manage the sources of contamination as they may contribute to the acquisition and spread of ESBL-Ec in humans and animals, thereby contributing to AMR.