Peri-implant inflammation contributes for loss of secondary stability of orthodontic mini-implants. The investigation of microbial colonization in this area would benefit its control, and consequently favor the long-term success of mini-implants. Therefore, the aim of this study was to determine the establishment and the evolution of microbial colonization process in orthodontic mini-implants for 3 months, since the time of their installation. One-hundred and fifty samples collected from 15 mini-implants were investigated from baseline up to 3 months. The biological material was obtained from peri-implant area using paper points. Nonspecific, Streptococcus spp, Lactobacillus casei and Candida spp colonizations were analyzed by cell growth methods. Porphyromonas gingivalis colonization was observed by 16S rDNA-directed polymerase chain reaction. Data from cell growth were submitted to the Wilcoxon sign rank test and results from molecular analysis were presented in a descriptive way. There was no significant difference in the microbial colonization among the examined time intervals, except for Streptococcus spp, between baseline and 24 h, which characterized the initial colonization in this time interval. Lactobacillus casei and Candida spp colonizations were insignificant. No Porphyromonas gingivalis was detected among the analyzed samples. The microbial colonization of mini-implants did not significantly change during the study. However, it should be monitored by orthodontists, since it is an important factor for mini-implants success.