Objective To assess the prevalence of unculturable bacteria in periapical abscess, radicular cyst, and periapical granuloma. Methods PubMed, Scopus, Science Direct, and Ovid databases were systematically searched from January 1990 to May 2020. All the included studies were cross-sectional design. The risk of bias was assessed using Joanna Briggs Institute check-list. Heterogeneity was described using meta-regression and mixed-effects model for lesion, country, and sequence technique moderators. Funnel plot and unweighted Egger’s regression test were used to estimate the publication bias. Microbiome data on diversity, abundance, and frequency of unculturable bacteria in the periapical lesions were reviewed, analysed, and the principal component analysis (PCA) was performed. Results A total of 13 studies out of 14,780, were selected for the final analysis. These studies focused on the prevalence of unculturable bacteria in periapical abscesses and related lesions. Approximately 13% (95% CI: 7–23%) of the cumulative number of bacteria derived from periapical abscesses was unculturable. Country moderator significantly (P = 0.05) affects the diversity summary proportion. While the pooled frequency of unculturable bacteria was 8%; 95% CI: 5, 14%, the estimate of the pooled abundance of unculturable bacteria was 5%; 95% CI: 2, 12% with a significant (P = 0.05) country moderator that affects the abundance summary proportion. Of the 62 unculturable bacteria, 35 were subjected to PCA and Peptostreptococcus sp. oral clone CK035 was the most abundant species in periapical abscesses. Hybridization techniques were found to be the most reliable molecular methods in detecting the abundance and frequency of unculturable bacteria. Conclusion The significant prevalence of unculturable bacteria in the periapical abscess, suggests that they are likely to play, a yet unknown, critical role in the pathogenesis and progression of the disease. Further research remains to be done to confirm their specific contributions in the virulence and disease progression.