Aims: The purposes of this in vitro study are to investigate the effect of instrumentation types (hand and rotary instrumentations) and obturation techniques (Cold lateral, and Thermafil) on vertical root fracture. Materials and Methods: The crown of the 100 single rooted teeth were removed leaving 12mm of the roots length, then the root randomly divided into four groups. Control-ve Group(n=10), Hand Group(n=30), ProTaper Group(n=30) and Helix Group(n=30) according to the type of the instrument used to prepared the root canals. After that, the last three groups subdivided into subgroups according to the obturation technique either by using Cold Lateral condensation technique or Thermafil obturation technique. After the sealer set, the roots were embedded in acrylic moulds and subjected to compressive loading, the load at which the fracture occurred was recorded and statically analysis. Results: the fracture resistance of the root prepared by Helix and ProTaper files were significantly greater than that obtained for the root canals prepared with hand K-files. The results of this study also demonstrated that when root canals obturated with Thermafil the roots resist the fracture load greater than that of the canals obturated with Cold Lateral condensation obturation but the difference is not statistically significant between them. Conclusions: the use of rotary nickel titanium files in endodontic treatment provides higher fracture resistance for the roots, and the use of the Cold Lateral condensation obturation technique may weaken the roots, so that the roots become more susceptible to the vertical root fracture.