Antimicrobial resistance is a major healthcare problem and a concern for global public health. In this current scenario, the use of non-antibiotic antimicrobials, such as heavy metals and biocides, has increased aiming to control the spread of antibiotic-resistant bacteria. Consequently, heavy metal tolerance genes (HMTG) and biocides tolerance genes (BTG) have been more frequently detected in Gram-negative bacilli. In this study, we searched for acquired HMTG, BTG and acquired antibiotic-resistance genes (ARG) and determined the minimum inhibitory concentration (MIC) of common heavy metals and biocides in multidrug-resistant and susceptible Gram-negative bacilli clinical isolates. High frequency of silA and pcoD genes was detected mainly among Klebsiella spp. and E. cloacae, regardless of their susceptible profile. merA gene was also found in isolates carrying silA-pcoD genes. ARG were detected in isolates that harbor silA and/or pcoD genes. BTG (qacΔE, ydgE, ydgF, mdfA, and emrE) were detected mostly in K. pneumoniae and E. cloacae isolates, regardless of their susceptible profile, and these isolates often co-harbored HMTG and/or ARG. Higher CuSO