Finding a new microbial source of transglutaminase (MTGase) and the study of the medium composition for MTGase production were the goals of this work. A total of 200 actinomycete-like strains were isolated from Brazilian soil samples and two of them named T10b and P20 were selected based on their ability to produce 0.15 U.mL-1 and 0.25 U.mL-1 of MTGase, respectively. Strain P20 was chosen to continue the study and was identified as Streptomyces sp. In order to optimize the MTGase activity, modifications of the usual media composition described for enzyme production were tested. The strategy adopted was: (1) screening experiment for the best carbon and nitrogen sources; (2) fractional factorial design (FFD) to elucidate the key ingredients in the media (the results indicated that the soybean flour, peptone, KH2PO4 and MgSO4.7H2O had a significant effect on MTGase) production and (3) central composite design (CCD) to optimize the concentration of the key components. The experimental results were fitted to a second-order polynomial model at the 95% level of significance (P < 0.05). Under the proposed optimized conditions, the model predicted a MTGase activity of 1.37 U.mL-1, very closely matching the experimental activity of 1.4 U.mL-1.