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Background: Conflicting findings on the potency of antibiotic pastes versus calcium hydroxide (CH) have been evident in the literature. Aims: To compare the antibacterial efficacy of single antibiotic paste (SAP), double antibiotic paste (DAP), triple antibiotic paste (TAP), and modified TAP (mTAP) with CH on bacterial biofilms. Methods: PubMed, Scopus, and Embase were comprehensively searched until August 23, 2021. The study protocol was registered in the PROSPERO. Ex vivo studies performed on Enterococcus faecalis or polymicrobial biofilms incubated on human/bovine dentin were selected. The quality of the studies was assessed using a customized quality assessment tool. Standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated for the meta-analysis. Meta-regression models were used to identify the sources of heterogeneity and to compare the efficacy of pastes. Results: The qualitative and quantitative synthesis included 40 and 23 papers, respectively, out of 1421 search results. TAP (SMD = −3.82; CI, −5.44 to −2.21; P < 0.001) and SAPs (SMD = −2.38; CI, −2.81 to − 1.94; P < 0.001) had significantly higher antibacterial efficacy compared to the CH on E. faecalis biofilm. However, no significant difference was found between the efficacy of DAP (SMD = −2.74; CI, −5.56–0.07; P = 0.06) or mTAP (SMD = −0.28; CI, −0.82–0.26; P = 0.31) and CH. Meta-regression model on E. faecalis showed that SAPs have similar efficacy compared to TAP and significantly better efficacy than DAP. On dual‑species (SMD = 0.15; CI, −1.00–1.29; P = 0.80) or multi‑species (SMD = 0.23; CI, −0.08–0.55; P = 0.15) biofilms, DAP and CH had similar efficacy. Conclusions: Ex vivo evidence showed that antibiotic pastes were either superior or equal to CH. The studied SAPs had considerably higher or similar antibacterial effectiveness compared to DAP, CH, and TAP. Hence, combined antibiotic therapy was not necessarily required for root canal disinfection ex vivo. [ABSTRACT FROM AUTHOR]