Trimethoprim-sulfamethoxazole is an active agent against Burkholderia pseudomallei and is being used in intensive and maintenance phases of melioidosis therapy. In this study, we evaluated the bactericidal activities of β-lactams (imipenem, ceftazidime and amoxicillin- clavulanate) alone and in combinations with trimethoprim-sulfamethoxazole against B. pseudomallei . Four clinical strains of B. pseudomallei were selected based on different genotypes that are frequently found in Malaysia. The minimum inhibitory concentrations of trimethoprim-sulfamethoxazole, ceftazidime, imipenem and amoxicillin-clavulanate were determined using microdilution broth method. The bactericidal activities and synergy effects of β-lactams and/or trimethoprim-sulfamethoxazole were evaluated by checkerboard and static time-kill analyses at 1×MIC concentration of each antibiotic. Using checkerboard method, the β-lactam/trimethoprim-sulfamethoxazole combinations exhibited ΣFIC of 0.75-4.00. In time-kill analysis, ceftazidime/trimethoprim-sulfamethoxazole combination demonstrated synergy against three strains (less 2.25-2.41 log 10 CFU/mL compared to the most active antibiotic monotherapy) whereas imipenem/trimethoprim-sulfamethoxazole combination regimen showed synergy against one strain (less 3.32 log 10 CFU/mL). No antagonist effect or major re-growth was observed in all combination regimens, whereas 11 out of 12 of β-lactam monotherapy regimens were associated with re-growth of bacteria. However, all β-lactam monotherapy regimens exhibited rapid and stronger killing activities against BUPS/07/14, in the initial 12 hours compared to β-lactam/ trimethoprim- sulfamethoxazole combination regimens. The combination of β-lactams with trimethoprim- sulfamethoxazole demonstrated better killing effect at 24 hours compared to monotherapy and no major bacterial regrowth was observed. Nevertheless, delay in killing activities of β-lactam/trimethoprim-sulfamethoxazole combination regimens against BUPS/07/14 need further examination because this phenomenon can lead to treatment failure in some patients.