Sodium hypochlorite (NaOCl), an effective endodontic irrigant against Enterococcus faecalis (EF), is harmful to periapical tissues. Natural pineapple-orange eco-enzymes (M-EE) and papaya eco-enzyme (P-EE) could be potential alternatives. This study aimed to assess the antimicrobial efficacy of M-EE and P-EE at different concentrations and fermentation periods against EF, compared to 2.5% NaOCl. Fermented M-EE and P-EE (3 and 6 months) at various concentrations were mixed with EF in a 96-well plate incubated for 24 h anaerobically. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of M-EE and P-EE were determined via EF growth observation. EF inhibition was quantitatively measured and compared between different irrigants using the one-way analysis of variance (ANOVA), and different fermentation periods using the independent-samples T-test. M-EE and P-EE showed MIC at 50% and MBC at 100% concentrations. There was no significant difference in antimicrobial effect when comparing M-EE and P-EE at 50% and 100% to 2.5% NaOCl. P-EE at 6 months fermentation exhibited higher EF inhibition compared to 3 months at concentrations of 25% (p = 0.017) and 0.78% (p = 0.009). The antimicrobial properties of M-EE and P-EE, at both 100% and 50% concentrations, are comparable to 2.5% NaOCl. They could therefore be potential alternative endodontic irrigants, but further studies are required.