Spiramycin is a macrolide antibiotic and antiparasitic that is used to treat toxoplasmosis and various other infections of soft tissues. In the current study, we evaluated the effects of α-cyclodextrin, β-cyclodextrin, or methyl-β-cyclodextrin supplementation to a synthetic culture medium on biomass and spiramycin production by Streptomyces ambofaciens ATCC 23877. We found a high stimulatory effect on spiramycin production when the culture medium was supplemented with 0.5% (w/v) methyl-β-cyclodextrin, whereas α-cyclodextrin or β-cyclodextrin weakly enhanced antibiotic yields. As the stimulation of antibiotic production could be because of spiramycin complexation with cyclodextrins with effects on antibiotic stability and/or efflux, we analyzed the possible formation of complexes by physical-chemical methods. The results of Job plot experiment highlighted the formation of a nonhost@guest complex methyl-β-cyclodextrin@spiramycin I in the stoichiometric ratio of 3:1 while they excluded the formation of complex between spiramycin I and α- or β-cyclodextrin. Fourier-transform infrared spectroscopy measurements were then carried out to characterize the methyl-β-cyclodextrin@spiramycin I complex and individuate the chemical groups involved in the binding mechanism. These findings may help to improve the spiramycin fermentation process, providing at the same time a new device for better delivery of the antibiotic at the site of infection by methyl-β-cyclodextrin complexation, as it has been well-documented for other bioactive molecules.