The increasing occurrence of multidrug-resistant Enterobacteriaceae in clinical and environmental settings has been seen globally as a complex public health challenge, mostly in the developing nations where they significantly impact on freshwater used for a variety of domestic purposes and irrigation. In this paper, we give details as regards the occurrence and antibiogram signatures of Enterobacteriaceae community in Tsomo and Tyhume rivers within the Eastern Cape Province, Republic of South Africa using standard methods. Average distribution of the presumptive Enterobacteriaceae in the rivers ranged from 1 10 2 CFU/100ml to 1.95 10 4 CFU/100ml. We confirmed 56 (70.8%) out of 79 presumptive Enterobacteriaceae isolated being species within the family Enterobacteriaceae through the Matrix-Assisted Laser Desorption Ionization Time of Flight technique. From this, Citrobacter -, Enterobacter -, Klebsiella species and Escherichia coli were selected (n=40) due to their pathogenic potentials for antibiogram profiling. The results of the antibiotic susceptibility testing gave a revelation that all the isolates were phenotypically multidrug-resistant while resistance against ampicillin (95%), tetracycline and doxycycline (88%) and trimethoprim-sulfamethoxazole (85%) antibiotics were most prevalent. The Multiple Antibiotic Resistance indices stretched from 0.22 to 0.94, with the highest index observed in a C. freundii isolate. Molecular characterisation using the PCR technique revealed the dominance of bla TEM (30%; 12/40) among the ten groups of extended-spectrum β-lactamase genes assayed. The prevalence of others was bla CTX-M genes including group 1, 2 and 9 (27.5%), bla SHV (20%), bla KPC (17.6%), bla GES (11.8%), bla IMP (11.8%), bla VIM (11.8%), bla OXA-1-like (10%), bla PER (2.5%), bla OXA-48-like (5.9%) and bla VEB (0%). From the six plasmid-mediated AmpC genes investigated bla ACC , bla EBC , bla FOX , bla CIT , bla DHA and bla MOX , only the first four were detected. In this category, the most dominant was bla EBC, with 18.4% (7/38). The prevalence of the non-β-lactamases include tetA (33.3%), tetB (30.5%) , tetC (2.8%), tetD (11.1%), tetK (0%), tetM (13.9%), catI (12%), catII (68%), sulI (14.3%), sulII (22.8%) and aadA (8.3%). Notably, a C. koseri harboured 42.8% (12/28) of the genes assayed for which includes five of the ESBL genes (including the only bla PER detected in this study), two of the pAmpC resistance genes ( bla ACC and bla CIT ) and five of the non-β-lactamase genes. To the best of our knowledge, this study gives the first report on C. koseri exhibiting co-occurrence of ESBL/AmpC β-lactamase genes from the environment. The detection of a bla PER producing Citrobacter spp. in this study is remarkable. These findings provide evidence that freshwater serves as reservoirs of antimicrobial resistance determinants which can then be easily transferred to human beings via the food chain and water.