The sarcotesta of Punica granatum fruit contains an antimicrobial lectin called PgTeL. In this work, we evaluated the antibacterial activity of PgTeL against five drug-resistant Escherichia coli isolates able to produce β-lactamases. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined by broth dilution. Morphometric and viability analyses were performed by flow cytometry, and ultrastructural changes were evaluated by scanning electron microscopy. Potential synergistic effects of PgTeL with antibiotics and anti-biofilm effect were also evaluated. PgTeL showed antibacterial activity against all isolates with MIC and MBC values ranging from 12.5 to 50.0 μg/mL and from 25.0 to 100.0 μg/mL, respectively. For most isolates, PgTeL postponed the growth start by at least ten hours. At the MIC, the lectin caused alterations in size, shape and structure of bacterial cells. The combination PgTeL-ceftazidime showed a synergistic effect for all isolates. Synergy was also detected with ampicillin (one isolate), carbenicillin (one isolate), cefotaxime (one isolate), cephalexin (four isolates) and cefuroxime (three isolates). PgTeL exhibited anti-biofilm activity against all isolates, causing ≥50% inhibition of biofilms at or above 6.25 μg/mL. The antibacterial effect of PgTeL and its synergy with antibiotics indicate that this fruit-derived molecule may have potential for future treatment of multidrug-resistant infections.