An important part of plant in vitro techniques is the sterilization of explants and the maintenance of aseptic conditions. Ideally, sterilizing materials should be effective on a vast range of microorganisms at low density. Nowadays, the use of compounds such as essential oils (EOs) and nanoparticles is applicable in microbiology studies. The main objective of this experiment was to study the substitution probability of silver nanoparticles (SNPs), thymol and carvacrol as novel sterilization agents in the tissue culture of Cynodon dactylon. Explants were sterilized with 70% ethanol for 2 min, and then 30% Clorox for 15 min. Sterilization complementary treatments (SNPs, thymol and carvacrol) were applied at different concentrations (100 and 200 mg l-1) with exposure times of 30, 60 and 120 min. According to the results, infection of bermudagrass explants was controlled successfully by SNPs, thymol and carvacrol. Examination of various concentrations in different exposure times showed that 200 mg L-1 SNPs in combination with 100 mg L-1 thymol in 60 min inhibited microbial growth. Thymol and carvacrol were more effective than SNPs in controlling bacteria and fungi contaminations. Finally, these novel agents could be used as an alternative to common chemical treatments for elimination and control of microbial population explants in in vitro conditions.