Antimicrobial resistance is a major health care problem as well as a concern for global public health. As a result, the use of nonantibiotic antimicrobials, such as heavy metals and biocides, has increased in a bid to control the spread of antibiotic-resistant bacteria. Consequently, heavy metal tolerance genes (HMTGs) and biocide tolerance genes (BTGs) have been more frequently detected in Gram-negative bacilli. In this study, we searched for acquired HMTGs, BTGs, and antibiotic resistance genes (ARGs) and determined the MICs of common heavy metals and biocides in multidrug-resistant and susceptible Gram-negative bacilli clinical isolates. A high frequency of silA and pcoD genes was mainly detected among Klebsiella spp. and Enterobacter cloacae regardless of their susceptible profile. The merA gene was also found in isolates carrying silA/pcoD genes. ARGs were detected in isolates that harboured silA and/or pcoD genes. BTGs (qacΔE, ydgE, ydgF, mdfA, and emrE) were mostly detected in Klebsiella pneumoniae and E. cloacae isolates regardless of their susceptibility profile, and these isolates often co-harboured HMTGs and/or ARGs. Higher copper sulphate MIC values were obtained under aerobic conditions, regardless of the presence or absence of pcoD and/or silA genes. Nevertheless, in most isolates carrying pcoD/silA, higher copper sulphate MIC values were determined under anaerobic conditions. Regarding AgNO