Nanomaterials (NMs) have found extensive commercial use in industries, healthcare and household applications however, their ecotoxicological effects remain elusive. Since, microbial communities play beneficial role in ecosystem like element cycling, bioremediation, nitrogen fixation, etc., effect of NMs over beneficial microbe’s physiology and viability remains to be studied in detail. Some beneficial microbe communities are severely affected by the release of NMs in the environment. Deinococcus radiodurans is known for its tolerance to oxidative stress caused due to irradiation. In this study, we have used metal, metal oxides, quantum dots (QDs) and carbon based NMs to assess their effect on the cell viability, uptake and ROS generation in D. radiodurans cells. The present study demonstrates in real-time by flow cytometry the internalization of different metal, metal oxide, QDs and carbon based NMs in D. radiodurans. Results show that all the tested NMs are significantly internalized in to the bacterial cells however, carbon based NMs exhibited highest internalization. Toxicity studies revealed that AgNPs exhibited maximum toxicity and reactive oxygen species (ROS) generation followed by QDs, CuO NPs and GO but, AuNPs and TiO2 NPs shows no toxic response in bacterial cells. The oxidative stress and uptake studies will provide insight about the mechanism of oxidative stress tolerance of D. radiodurans.