In vitro propagation is a technique that has been used to produce large-scale, healthy, pathogen-free plants. However, the presence of endophytic microorganisms in explant tissues is one of the limiting factors for the in vitro establishment of any species. In this sense, we studied the effectiveness of PPM® biocide (Plant Preservative Mixture) and Chlorothalonil fungicide to control and/or reduce microbial and fungal contamination in mature nodal segments of different Persian lime clones under in vitro conditions. The results showed that explants cultured in MS medium (Murashige and Skoog 1962) supplemented with 0.2 mL L-1 of PPM biocide + 1.0 mL L-1 of Chlorothalonil were adequate to achieve a higher percentage of disinfection (44%) in mature nodal segments in the Persian lime clones. Furthermore, it was observed that PPM concentrations above 0.3 mL L-1 resulted in greater phytotoxicity in mature nodal segments at 60 days.