This systematic review assessed the effect of different root canal instrumentation systems on endotoxin reduction from the root canal system. The literature search was conducted in two electronic databases (PubMed and Scopus) using specific key words. The search strategy followed the PRISMA guidelines. Qualitative synthesis and quantitative synthesis of the data were performed and data interpretation was done based on the guidelines in the Cochrane Handbook. The risk of bias was assessed using Cochrane criteria. The initial search yielded 600 citations, of which three papers met the criteria for inclusion in this review. Studies showed a significant reduction in endotoxin following rotary and reciprocating instrumentation, with no significant differences between them. The meta-analysis showed no statistical significant difference between reciprocation and rotary files (P > 0.05). In conclusion, the instrumentation techniques compared in this review decrease endotoxin content from root canals, with no significant difference between them.