The aim of this study was to establish the antimicrobial activities of S-(3,4-dichlorobenzyl)isothioureahydrochloride (A22) and a series of structurally related compounds against multidrug-resistant (MDR)bacteria. The minimum inhibitory concentrations (MICs) of 21 compounds were determined against 18 strains of pathogenic bacteria in addition to Pseudomonas aeruginosa (n = 19) and Burkholderia cepaciacomplex (BCC) (n = 20) isolated from the sputa of cystic fibrosis patients. Selected compounds were tested against further isolates, including P. aeruginosa (n = 100), BCC (n = 12) and Stenotrophomonas maltophilia(n = 19). The interaction of S-(4-chlorobenzyl)isothiourea hydrochloride (C2) in combination with conventional antimicrobials was examined against 10 P. aeruginosa strains. Selected compounds were also tested against Enterobacteriaceae producing NDM-1 carbapenemase (n = 64) and meticillin-resistant Staphylococcus aureus (MRSA) (n = 37). Of the 21 compounds, 14 showed antimicrobial activity that was generally more pronounced against Gram-negative bacteria. Against P. aeruginosa, the most active compound was C2 [MIC for 50% of the organisms (MIC50) = 32 g/mL]. This compound was also the most activea gainst BCC, with all isolates inhibited by 64 g/mL. For all ten strains of P. aeruginosa subjected to combination testing with C2 and conventional antimicrobials, a bactericidal effect was achieved with at least one combination. C2 and A22 both showed strong activity [MIC for 90% of the organisms (MIC90) = 4 g/mL]against Enterobacteriaceae that produced NDM-1 carbapenemase. Finally, S-(4-chlorobenzyl)-N-(2,4 dichlorophenyl)isothiourea hydrochloride showed good activity (MIC90 = 8 g/mL) against MRSA. This work establishes the activity of isothiourea derivatives against a broad range of clinically important MDR bacteria.