This study aimed to evaluate the efficacy of intracanal Enterococcus faecalis reduction using pediatric rotary (EndoArt Pedo Kit Blue, EasyInSmile X-Baby and Denco Kids), rotary (ProTaper Next) and reciprocating (WaveOne Gold) file systems through microbiological analyses in primary molars. Seventy-five mandibular primary second molars were selected and divided into five instrumentation groups and a negative control group. After incubation, five roots were used to confirm biofilm formation on the root canals. Before and after instrumentation, bacterial samples were collected. The bacterial load reduction was statistically analyzed by using Kruskall-Wallis and Dunn post hoc tests at a significance level of 0.05. Denco Kids and EndoArt Pedo Kit Blue promoted higher bacterial reduction than EasyInSmile X-Baby systems. There was no difference in bacterial reduction between ProTaper Next rotary file systems and other groups. Among the single-file techniques, instrumentation with the Denco Kids rotary system showed a more significant bacterial load reduction than WaveOne Gold (p < 0.05). All systems used in the study reduced bacterial counts from root canals in primary teeth. Further studies are required to generate more information about the use of pediatric rotary file systems in clinics.