Despite the huge and efficient functionalities of reduced graphene oxide (RGO) for bioengineering applications, the use of harsh chemicals and unfavorable techniques in their production remains a major challenge. Microbial production of reduced graphene oxide (RGO) using specific bacterial strains has gained interest as a sustainable and efficient method. The reduction of GO to RGO by selected bacterial strains was achieved through their enzymatic activities and resulted in the removal of oxygen functional groups from GO, leading to the formation of RGO with enhanced structural integrity. The use of microorganisms offers a sustainable approach, utilizing renewable carbon sources and mild reaction conditions. This study investigates the production of RGO using three different bacterial strains: (), (), and () and evaluates its toxicity for safe utilization. The aim is to assess the quality of the produced RGO and evaluate its toxicity for potential applications. Thus, this study focused on the microbial production of reduced graphene oxides well as the investigation of their cellular interactions. Graphite-derived graphene oxide was used as a starting material and microbially reduced GO products were characterized using the FTIR, Raman, XRD, TGA, and XPS methods to determine their physical and chemical properties. FTIR shows that the epoxy and some of the alkoxy and carboxyl functional groups were reduced by and , whereas the alkoxy groups were mostly reduced by . The / ratio from Raman spectra was found as 2.41 for GO. A substantial decrease in the ratio as well as defects was observed as 1.26, 1.35, and 1.46 for ERGO, LLRGO, and LPRGO after microbial reduction. The XRD analysis also showed a significant reduction in the interlayer spacing of the GO from 0.89 to 0.34 nm for all the reduced graphene oxides. TGA results showed that reduction of GO with provided more reduction than other bacteria and formed a structure closer to graphene. Similarly, analysis with XPS showed that L lactis provides the most effective reduction with a C/O ratio of 3.70. In the XPS results obtained with all bacteria, it was observed that the C/O ratio increased because of the microbial reduction. Toxicity evaluations were performed to assess the biocompatibility and safety of the produced RGO. Cell viability assays were conducted using DLD-1 and CHO cell lines to determine the potential cytotoxic effects of RGO produced by each bacterial strain. Additionally, apoptotic, and necrotic responses were examined to understand the cellular mechanisms affected by RGO exposure. The results indicated that all the RGOs have concentration-dependent cytotoxicity. A significant amount of cell viability of DLD-1 cells was observed for reduced graphene oxide. However, the highest cell viability of CHO cells was observed for reduced graphene oxide. All reduced graphene oxides have low apoptotic and necrotic responses in both cell lines. These findings highlight the importance of considering the specific bacterial strain used in RGO production as it can influence the toxicity and cellular response of the resulting RGO. The toxicity and cellular response to the final RGO can be affected by the particular bacterial strain that is employed to produce it. This information will help to ensure that RGO is used safely in a variety of applications, including tissue engineering, drug delivery systems, and biosensors, where comprehension of its toxicity profile is essential.