The study aimed to evaluate the efficiency of the Brush File and "passive ultrasonic" irrigation as irrigation agitation techniques compared to "conventional syringe irrigation" on "biofilm" eradication and "calcium hydroxide paste" removal from straight root canals. Materials and Methods: Thirty-six extracted human single-rooted teeth with single canals were decoronated, mechanically prepared, and inoculated with "E. faecalis" for three weeks, and biofilm formation was verified using SEM (n = 3). According to the irrigation agitation method, teeth were divided into three equal groups (n = 11); Brush File group, PUI group, and "conventional syringe irrigation" group "control group". Residual bacterial biofilm was taken by three sterile paper points, and CFUs were counted and calculated to CFUs/ml. For comparing calcium hydroxide removal efficacy, another thirty-three extracted single-rooted with single canals were instrumented, filled with Ca(OH)2 paste for one week, and assigned randomly to one of three groups (n = 11), as previously mentioned. Then, Specimens were longitudinally split, and "Ca (OH)2 remnants" were examined under 25x magnification using a stereomicroscope, calculated in mm 2 , and the percentage of Ca(OH)2 in the specimen's "coronal, middle, and apical thirds" was recorded using image analysis software. Results: Regarding "E. faecalis" biofilm eradication, there was a "significant difference" (p= 0.00) between the three groups; however, there was "no significant difference" between the Brush File and PUI. Regarding Ca(OH)2 removal, the intergroup comparison revealed a "significant difference" across the three groups at "coronal, middle, and apical thirds" (p= 0.00), with "no significant difference" between the Brush File and PUI groups. The Brush File and CSI groups had "significantly higher "values of " Ca(OH)2 remnants" in the apical third. Conclusion: Brush File was as efficient as PUI in terms of "biofilm eradication" and "calcium hydroxide" removal, and both agitation techniques were superior to the "conventional irrigation" regimen.