The objective of this systematic review and meta-analysis was to evaluate the effect of periodontal surgery on the subgingival microbiome. Periodontitis is a chronic inflammation of the tooth supporting tissues caused by the dysbiosis of the subgingival biofilm. It is managed through different non-surgical and surgical treatment modalities. Recent EFP S3 guidelines recommended performing periodontal surgery as part of Step 3 periodontitis treatment after Step 1 and Step 2 periodontal therapy, with the aim to achieve pocket closure of persisting sites. Changes in the sub-gingival microbiome may explain the treatment outcomes observed at different time points. Various microbiological detection techniques for disease-associated pathogens have been evolved over time and have been described in the literature. However, the impact of different types of periodontal surgery on the subgingival microbiome remains unclear. A systematic literature search was conducted in Medline, Embase, LILACS and Cochrane Library supplemented by manual search (23DEC2019, updated 21APR2022). From an initial search of 3046 studies, 28 were included according to our specific inclusion criteria. Seven microbiological detection techniques were used to analyse disease-associated species in subgingival plaque samples: optical microscope, culture, polymerase chain reaction (PCR), checkerboard, enzymatic reactions, immunofluorescence and 16S gene sequencing. The included studies exhibited differences in various aspects of their methodologies such as subgingival plaque sample collection or treatment modalities. Clinical data showed a significant decrease in probing pocket depths (PPD) and clinical attachment loss (CAL) after periodontal surgery. Microbiological findings were overall heterogeneous. Meta-analysis was performed on a sub-cohort of studies all using checkerboard as a microbiological detection technique. Random effect models for Treponema denticola (T.denticola), Porphyromonas gingivalis (P.gingivalis) and Tannerella forsythia (T.forsythia) did not show a significant effect on mean counts 3 months after periodontal surgery. Notably, Aggregatibacter actinomycetemcomitans (A.actinomycetemcomitans) showed a significant increase 3 months after periodontal surgery. 16S gene sequencing was used in one included study and reported a decrease in disease-associated species with an increase in health-associated species after periodontal surgery at 3 and 6 months. This systematic review has shown that the effect of periodontal surgery on the changes in subgingival microbiome is heterogeneous and may not always be associated with a decrease in disease-associated species. The variability could be attributed to the microbiological techniques employed for the analysis. Therefore, there is a need for well-designed and adequately powered studies to understand how periodontal surgery influences the subgingival microbiome and how the individual's microbiome affects treatment outcomes after periodontal surgery.