Background: The qacE gene and its variant, qacE∆1, have been associated with resistance to antimicrobials and biocides. This poses a threat to infection prevention, control and treatment. Several studies investigated this relationship with conflicting results. The aim of this research was to determine the prevalence of qacE and qacE∆1 in clinical extended spectrum β-lactamase- (ESBL) and carbapenemase-producing Klebsiella spp. and Enterobacter spp. and elucidate the association of these genes with resistance to sodium hypochlorite. Methods: This study included 216 isolates of ESBL- and carbapenemase-producing multidrug-resistant (MDR) Klebsiella spp. and Enterobacter spp. These isolates were identified by VITEK-2 analyser. The MIC and MBC of sodium hypochlorite were determined using the microbroth serial-dilution method. PCR was used to detect gene variants. A regression analysis investigated any association between qacE genotypes, MIC and MBC, as well as antimicrobial drug resistance profiles. Results: Overall, there was a high prevalence of qacE and qacE∆1 variants (84.7%; 95% CI, 79.2–89.2). There was a high prevalence of qacE∆1 (80.6%; 95% CI, 74.6–85.6) as compared to qacE (15.3%, 95% CI, 10.8–20.8). The MIC₅₀ and MIC₉₀ of the isolates ranged between 7031 mg/L and 9375 mg/L and 14,060 mg/L and 18,750 mg/L, respectively, while the MBC ranged from 48,750 mg/L to 18,750 mg/L. There was no association between qacE genotypes and high MIC and MBC as well as antimicrobial drug resistance. Conclusions: The MIC and MBC of sodium hypochlorite are higher than what is currently used for disinfection in Botswana. There is a high prevalence of qacE and qacE∆1; however, these genes do not seem to be associated with resistance to sodium hypochlorite.