Decentralized wastewater treatment systems afford a unique opportunity to implement a One Water paradigm through water reuse and resource recovery. Simultaneously, poorly managed on-site wastewater treatment systems (OWTSs) pose significant threats to human health, although their role in environmental antimicrobial resistance (AMR) is understudied. We deployed culture-based methods adapted from the World Health Organization’s Tricycle Protocol to assess the potential role of OWTSs in discharging third generation cephalosporin-resistant E. coli (3GCR-Ec) and extended-spectrum beta lactamase-producing E. coli (ESBL-Ec). We detected 3GCR-Ec in 74% of ATU effluents (geometric mean of 440 CFU/100 mL) and ESBL-Ec in 19%. E. coli isolates in these effluents were resistant to an average of 4.11 antibiotic compounds. ESBL-Ec were detected in up to 19% of surface water samples from rivers known to be impacted by OWTS discharges. Human sewage marker abundance was significantly higher in samples containing 3GCR-Ec (p = 0.0026) and ESBL-Ec (p = 0.0121) and the proportion of samples positive for 3GCR-Ec was strong correlated with river discharge percentile (r > 0.67, p < 0.0292). Our findings emphasize the unique human-environment interface of decentralized wastewater treatments systems must be carefully considered in both One Health and One Water paradigms to safely and sustainably meet our needs.