Inclusion complexation of rifampicin (RIF) with several types of cyclodextrins (beta CD, hydroxypropyl-beta CD, gamma CD, hydroxypropyl-gamma CD) in aqueous solutions at different pH values was investigated to assess the interactions between RIF and cyclodextrins (CDs). Molecular modeling was performed to determine the possible interactions between RIF and CDs at several pH values. The inclusion complexes were characterized by differential scanning calorimetry, Fourier transform infrared spectroscopy, powder X-ray diffractometry, and scanning electron microscopy. Moreover, this study evaluated the dissolution profile and antibacterial activity of the formed complexes. Phase solubility analysis suggested the formation of RIF-CD affirmed 1:1 stoichiometry at all pH values (except RIF-beta CD at pH 4.0 and both beta CD and gamma CD at pH 9.0). The inclusion complexation of RIF with CD successfully increased the percentage of RIF released in in vitro studies. The inclusion complexes of RIF exhibited more than 60% of RIF released in 2 h which was significantly higher (p < 0.05) than release of pure RIF, which was only less than 10%. Antibacterial activity of RIF-CD complexes (measured by the minimum inhibitory concentration of RIF against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus) was lower for both RIF-beta CD and RIF-HP gamma CD at pH 7.0 to pure RIF suspension. In conclusion, this work reports that both beta CD and gamma CD can be used to enhance the solubility of RIF and thus, improve the effectivity of RIF by decreasing the required daily dose of RIF for the treatment of bacterial infections.