Certain environmental variables are responsible for the survival of microorganisms in aquatic environments. The influence of these environmental factors in each season (winter, autumn, spring and summer) of the year can be used to track changes in a microbial population in freshwater resources. In this study, we assessed the effect of seasonal shifts in environmental variables including temperature, pH, total dissolved solids (TDS), total suspended solids (TSS), biochemical oxygen demand (BOD) and turbidity (TBS) among others on the density of Acinetobacter species in the Great Fish, Keiskamma and Tyhume rivers in the Eastern Cape Province, South Africa. Water samples and values of the environmental factors were taken from the rivers for 12 months. The density of presumptive Acinetobacter species was estimated from the culture of water samples on a CHROMagar selective medium, while the Acinetobacter-specific recA gene was targeted for the identification of Acinetobacter species using PCR assay. The multivariate relationship between seasons and changes in variables was created using PCA, while the effect of seasonal shifts in the environmental variables on the density of Acinetobacter species was evaluated using correlation test and topological graphs. Positive association patterns were observed between the seasons, environmental factors and the bacterial density in the rivers. In addition, temperature, TBS, TSS and BOD tended to influence the bacterial density more than other physicochemical factors in the rivers across the seasons. Of the total 1107 presumptive Acinetobacter species, 844 were confirmed as Acinetobacter species. Therefore, these findings suggested that the rivers contain Acinetobacter species that could be useful for basic and applied study in ecology or biotechnology, while their clinical relevance in causing diseases cannot be underestimated.