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 freshwater used for a variety of domestic purposes and irrigation. This paper detail the occurrence and antibiogram signatures of the Enterobacteriaceae community in Tsomo and Tyhume rivers within the Eastern Cape Province, the Republic of South Africa, using standard methods. The average distribution of the presumptive Enterobacteriaceae in the rivers ranged from 1 × 102 CFU/100ml to 1.95 × 104 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. 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. The 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 blaTEM (30%; 12/40) among the eight groups of β-lactams resistance determinants assayed. The prevalence of others was blaCTX-M genes including group 1, 2 and 9 (27.5%), blaSHV (20%), blaOXA-1-like (10%), blaPER (2.5%), and blaVEB (0%). The frequencies of the resistance determinants for the carbapenems were blaKPC (17.6%), blaGES (11.8%), blaIMP (11.8%), blaVIM (11.8%), and blaOXA-48-like (5.9%). Out of the six plasmid-mediated AmpC (pAmpC) genes investigated blaACC, blaEBC, blaFOX, blaCIT, blaDHA, and blaMOX, only the first four were detected. In this category, the most dominant was blaEBC, 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 (22.9%) 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 blaPER detected in this study), two of the pAmpC resistance genes (blaACC and blaCIT), and five of the non-β-lactamase genes. This study gives the first report on C. koseri exhibiting the co-occurrence of ESBL/AmpC β-lactamase genes from the environment to the best of our knowledge. The detection of a blaPER 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.