Objectives This systematic review and meta-analysis aimed to assess the prevalence of Bacteroidetes, Spirochaetes, and Fusobacteriales in symptomatic versus asymptomatic apical periodontitis as a primary objective. The secondary objective was to evaluate the prevalence of these species independently in symptomatic and asymptomatic apical periodontitis. Materials and methods An electronic search of the PubMed, Scopus, and Open-Grey databases was carried out from November 2022 to February 2023 and was later updated through July 2024..The risk of bias was assessed using the New Castle Ottawa scale. The quality of evidence was assessed using the Grading of Recommendations. Assessment, Development, and Evaluation. Results The initial search resulted in 968 records. Following the removal of duplicates and a review of titles and abstracts, 66 studies underwent full-texts analysis. Twenty studies were deemed eligible for inclusion. For the first outcome, a fixed-effects model was used. In a total of 7 studies with 193 participants, Spirochaetes were more prevalent in symptomatic apical periodontitis (p < .05) with a risk ratio of 1.91 [ 95% CI 1.25-2.92]. No significant difference was observed (p'.05) in the prevalence of Bacteroidetes (12 studies with 451 participants) and Fusobacteriales (7 studies with 205 participants) in symptomatic vs asymptomatic apical periodontitis with a risk ratio of (.96), and (1.1), [95% CI, .78-1.19], and [95% CI, 0.84.1.44], respectively. The overall quality of evidence was low. Conclusions The confirmation of the prevalence of Bacteroidetes and Fusobacteriales in symptomatic vs asymptomatic apical periodontitis remains uncertain. However, there is an association between Spirochetes and symptomatic apical periodontitis. Additional research is required to address the limitations of the current body of evidence. Clinical relevance Identifying key pathogens in symptomatic apical periodontitis can help develop targeted interventions that address the underlying microbial causes.