Burkholderia spp. are opportunistic human pathogens that infect persons with cystic fibrosis and the immunocompromised. Burkholderia spp. express class A and C β-lactamases, which are transcriptionally regulated by PenRA through linkage to cell wall metabolism and β-lactam exposure. The potency of temocillin, a 6-methoxy-β-lactam was tested against a panel of multidrug resistant (MDR) Burkholderia spp. In addition, the mechanistic basis of temocillin activity was assessed and compared to ticarcillin. Susceptibility testing with temocillin and ticarcillin was conducted, as well as biochemical analysis of the PenA1 class A β-lactamase and AmpC1 class C β-lactamase. Molecular dynamics simulations (MDS) were performed using PenA1 with temocillin and ticarcillin. The majority (86.7%) of 150 MDR Burkholderia strains were susceptible to temocillin, while only 4% of the strains were susceptible to ticarcillin. Neither temocillin nor ticarcillin induced bla expression. Ticarcillin was hydrolyzed by PenA1 (kcat/Km = 1.7±0.2 μM-1s-1), while temocillin was slow to form a favorable complex (Kiapp = ~2 mM). Ticarcillin and temocillin were both potent inhibitors of AmpC1, with Ki app values of 4.9±1.0 μM and 4.3±0.4 μM, respectively. MDS of PenA revealed that ticarcillin is in an advantageous position for acylation and deacylation. Conversely, with temocillin, active site residues K73 and S130 are rotated and the catalytic water molecule is displaced, thereby slowing acylation and allowing the 6-methoxy of temocillin to block deacylation. Temocillin is a β-lactam with potent activity against Burkholderia spp. as it does not induce bla expression and is poorly hydrolyzed by endogenous β-lactamases.