Simultaneous loading of hydrophobic and hydrophilic drugs into a drug delivery system is a difficult task and challenges still remain due to opposite nature of drugs. So far, most of the drug delivery systems have been designed based on liposomes, dual emulsions or porous nano-silica cavities. The application of Graphene and its oxidized derivatives as nanocarriers (NCs) have grown rapidly in the past few years. The first part of this study, using molecular modeling methods, provides details on the possibility and the mechanism of simultaneous loading of two hydrophobic and hydrophilic drugs, Rifampicin and Isoniazid, into graphene oxide (GO). The results confirmed the possibility of simultaneous loading of drugs in GO. The binding energies, calculated at the B3LYP-D3/6-31G(d) level of theory, are: −46.5 and −14.0 kJ mol−1 for Isoniazid and Rifampicin, respectively. Drugs loading, as also evidenced in the second part of study experimentally. The drug-loaded NCs were coated with biopolymers of Chitosan and Gum Tragacanth. SEM results confirmed that GO-NCs have produced with a diameter < 100 nm. Also, the results showed that the final diameters of coated NCs fall in the range 120–130 nm. A loading of 42.7% and 22% was also achieved for Rifampicin and Isoniazid, respectively. The release of Isoniazid reaches 93% after 60 h, and for Rifampicin is 88% after 72 h. To determine the toxicity of the biopolymer coated NCs, MTT test was used. Also, the antimicrobial efficacy of coated NCs loaded with drugs was evaluated versus pure drugs against Mycobacterium tuberculosis. The results showed that the drug co-loaded NCs have the same efficacy as pure drugs in their MIC concentrations.