Aims and objectives: Evaluating the antimicrobial efficacy of the novel combinations of zinc oxide mixed with ajwain oil (ZNOA) and combination of ajwain and eugenol (ZNOAE) vs conventionally used zinc oxide eugenol (ZNOE) against endodontic pathogens like Escherichia coli and Enterococcus faecalis. Materials and methods: The pure cultures of E. coli (MTCC 443) and E. faecalis (MTCC 439) were revived and grown on selective cultural media. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the test materials were determined correspondingly through sequential dilution and agar well diffusion methods, as per Clinical and Laboratory Standard Institute (CLSI) guidelines. The data values were presented as mean ± standard deviation (SD). The comparisons among groups were completed through the one-way analysis of variance (ANOVA) test, whereas intragroup pairwise comparisons were completed using the unpaired t-test (p < 0.05). Results: Minimum inhibitory concentration values against E. coli and E. faecalis of ZNOE were 250 and 500 µg/mL, ZNOA was 250 µg/mL, and ZNOAE were 125 and 250 µg/mL, correspondingly. MBC values in the form of inhibition zone against E. coli by ZNOE were 21.33 ± 1.53 mm, ZNOA 18.67 ± 1.53 mm, and ZNOAE 20.33 ± 1.53 mm. The E. faecalis inhibition zone for ZNOE was 14.33 ± 2.08 mm, ZNOA 18.67 ± 2.08 mm, and ZNOAE 24.33 ± 1.53 mm. Conclusion: All test materials demonstrated good antibacterial efficacy. However, between the novel combinations of test materials, ZNOA showed better antimicrobial efficacy against resistant endodontic pathogens than ZNOE.