Objective: Cloning ornamental plants, particularly chimeric varieties, is a crucial strategy for enhancing competitiveness in the ornamental plant market. Since the only method to propagate clones of chimeric Sansevieria varieties is through offset production from rhizome origins, optimizing sterilization techniques for in vitro rhizome explants is imperative. A significant drawback of plant tissue culture is the risk of microbial contamination, making the removal of contaminants a foundational step for successful tissue culture. Initial experiments on Sansevieria trifasciata rhizome explants revealed that conventional disinfectants were ineffective at eliminating contamination. Consequently, this study aimed to improve culture conditions by evaluating various factors essential for successful plant tissue culture.Methods: In a preliminary experiment, two types of mother-plant growing media (perlite and conventional greenhouse soil) were compared using a t-test with ۵۰ experimental units within a group. The second experiment was conducted on perlite as the mother-plant growing medium using a factorial arrangement with three factors based on the completely randomized design with three replications. The factors included three rhizome explant types (single node, terminal meristem, and rhizomes with terminal buds), benomyl and carbendazim fungicides at two concentrations of ۲% and ۱۰%, and mercuric chloride as the chemical disinfectant at two concentrations of ۰.۱% and ۰.۲% for ۲ and ۲۰ minutes plus a control without mercuric chloride.Results: Results indicated that culturing mother plants in the perlite bed significantly reduced microbial loads. The results of the second experiment showed that treatment with ۱۰% benomyl fungicide effectively diminished microbial contamination. Among the disinfectants tested, a ۲۰-minute treatment of explants with ۰.۲% mercuric chloride resulted in the lowest fungal contamination. Among the explant types, single-node explants exhibited the least fungal contamination.Conclusion: pre-treating single-node rhizome explants with ۱۰% benomyl fungicide, followed by a ۲-minute exposure to ۷۰% alcohol and a ۲۰-minute treatment with ۰.۲% mercuric chloride in a laminar flow cabinet, leads to optimal microbial decontamination. These findings underscore the importance of specific sterilization protocols in enhancing the efficacy of plant tissue culture methods for chimeric Sansevieria propagation.