Purpose: The aim of this study was to explore the surface roughness and hardness of the implant head of orthodontic mini-implants made from different alloys before and after their in vitro exposure to agents for prevention of gingivitis, mucositis, and peri-implantitis: chlorhexidine and probiotics. Materials and Methods: Three types of commercially available mini-implants were tested: 316 stainless steel, titanium Grade 5, and titanium Grade 23 (both Ti-6Al-4V alloys with the same atomic weight percentage of Ti, Al, and V, with the difference being in maximal reduction of O2 in Grade 23 to 0.13% of atomic weight). They were immersed in three experimental solutions: artificial saliva, saliva with probiotic bacteria Lactobacillus reuteri, and saliva with oral antiseptic chlorhexidine (CHX). Samples were immersed for 28 days, thermocycled, then stored in an incubator at 37°C. Surface roughness and microhardness on five samples of each of the three implant types were measured by atomic force microscopy and the Vickers method, respectively. Results: Exposure of titanium implant Grade 5 to probiotics significantly increased roughness compared with other media (P .005). Exposure to CHX significantly increased the roughness of steel implants (P .05). Neither saliva, probiotic, nor CHX altered microhardness of titanium implants significantly. In steel implants, the exposure to CHX and probiotics decreased microhardness compared with unexposed implants (P .031), but not in comparison to saliva. Conclusion: Probiotics seem to increase roughness of titanium mini-implants, while CHX seems to increase roughness of steel mini-implants. Only stainless steel implants had an altered, decreased hardness after exposure to CHX, although the same was found after their exposure to saliva. For patients undergoing orthodontic treatment with temporary anchorage units, CHX could be recommended for titanium, and probiotics for stainless steel mini-implants in oral-hygiene maintenance. Keywords: biomaterials, biomechanics, microbiology