For the comprehensive determination of water quality in the Akaki River various physicochemical characteristics were analyzed in this study with various methods: in-situ and ex-situ measurements and citizen science was used to determine nutrients, heavy metals, fecal coliforms, fecal streptococci, ESBL resistant total coliform and fecal coliform bacteria and the river water microbial community composition with molecular methods. The molecular analysis of environmental DNA used a low-cost, portable high throughput sequencer (MinION) to comprehensively characterize the overall microbial water quality in the Akaki River by 16S rRNA gene amplicon sequencing. qPCR quantification of 16S rRNA genes further enabled to estimate the absolute abundances of bacterial community members identified with MinION sequencing. The MinION results were compared with qPCR assays for Arcobacter butzleri, Vibrio cholerae, Bacteroides dorei and Human E coli, and the comparison validated the reliability of the MinION results for these pathogens. MinION analysis identified the potential role of bacterial genera not conventionally monitored, Arcobacter, Acinetobacter and Aeromonas, as predominant fecal pollution indicators/waterborne hazards. Complementary quantitative PCR analysis validated the high Arcobacter butzleri abundances observed in the urban influenced Akaki River water samples. Fecal Coliform (FC) and Fecal Streptococci counts in all the water samples exceeded CCME guidelines for irrigation water (i.e. 100 CFU/100 ml, CCME, 2008). The levels of these two indicator organisms varied along the river catchment, with lowest counts in the most upper stream sites and highest counts in the mid and downstream sites. The highest abundance of fecal coliform bacteria were detected in sampling sites 03 (Kebena) and SS 06 (little Akaki); 1,150,000 and 1,075,000 CFU in 100 mL respectively. Highest abundance streptococci were marked at SS 02; 85,500 CFU in 100 mL. The Akaki river and Kality wastewater treatment plant (WWTP) samples had prevalence of antibiotic resistance bacteria; namely ESBL producing total coliform (TC) and FC bacteria which can resists third generation cephalosporin antibiotics such as ceftazidime, cefotaxime, and ceftriaxone and also resist aztreonam and fluconazole treatments. The highest abundance of ESBL producing Fecal Coliform and Total Coliform bacteria were found in the WWTP influent and at sampling site SS 03 in the Kebena River which flows through central Addis Ababa. The average ESBL resistant Fecal Coliform and Total Coliform results of the WWTP influent and SS 03 (Kebena) was 6.01, 6.92 and 4.62, 5.96 LOG10 units of CFU/100 ml, respectively. Moreover the results showed that wastewater discharge made a significant contribution for the elevated AMR bacteria in surface water. Physicochemical parameters pH and iv rntemperature of the Akaki River were within the agreed limits of USEPA standards. Whereas the electric conductivity value at sampling site SS 06 (Little Akaki) and SS 03 (Kebena) exceeded the Ethiopian standards. Nitrite, ammonia and phosphate concentrations, except for the upstream site and Ambissa exceeded USEPA standard limits, and DO were below the standard requirements. From all the measured heavy metal parameters, the concentration of arsenic in the Big Akaki River surpassed the standard limit of the WHO guideline for drinking water, but was within the agreed limit of CCME guidelines for irrigation water. This indicates that the Big Akaki catchment in the study area was not exposed to industrial pollution which may be a potential source of heavy metals. Citizen Science data of Akaki river pH, nitrite, nitrate, ammonia, and alkalinity concentrations were in line with laboratory analysis data and provide reasonable indications of water quality.