Bacterial resistance to conventional antibiotics is a serious challenge that requires novel antibacterial agents. Moreover, wastewater from dairy farms might contain countless number of pathogens, organic contaminants and heavy metals that consider a threat to the terrestrial and aquatic environment. Therefore, the development of cost-effective, highly operation-convenient, recyclable multifunctional antimicrobial agents became an urgent necessity. Layered double hydroxides (LDH) have shown promising results as antibacterial agents. However, more work is required to further investigate and improve the antimicrobial performance of LDH structures against pathogens. In this study three Zn–Fe based LDH were investigated for real dairy wastewater disinfection. The three LDH samples were cobalt substituted Zn–Fe LDH (CoZnFe), magnesium substituted Zn–Fe LDH (MgZnFe) and MgZnFe-Triazol LDH (MgZnFe-Tz) nanocomposite. Seventy-five wastewater samples were collected from a dairy farm sewage system. The sensitivity of isolated pathogens was tested against two commonly used disinfectants (Terminator and TH4) and was assessed against the three LDH samples at different concentrations. The overall prevalence of S.agalactiae, S.dysgalactiaeandStaph. aureuswas significantly at 80.0% (P-value = 0.008, X2 = 9.700). There was variable degree of resistance to the tested disinfectants, whereas the antimicrobial activity of CoZnFe LDH was increased significantly at a concentration of 0.005 mg/L followed by MgZnFe LDH while MgZnFe-Tz LDH showed minor antibacterial potency. It was concluded that CoZnFe LDH showed a better biocidal activity in killing the isolated resistant pathogens, making it a good choice tool in combating the zoonotic microbes in wastewater sources.