Background: Klebsiella pneumoniae is a major cause of hospital-acquired infections in Jamaica. Objective: We aimed to determine their antimicrobial resistance profiles and to assess biofilm formation in the presence of antibiotic, nicotine and amino acid starvation stresses. Methodology: Antimicrobial susceptibility and multiple antimicrobial resistance (MAR) index were determined for 23 K. pneumoniae strains. Biofilm production was evaluated in the presence of 50 μg/ml ceftazidime or gentamicin, 0–4 mg/ml nicotine, or 0.5 mg/ml serine hydroxamate (to induce amino acid starvation). Genetic relatedness, and the presence of type 3 fimbriae (mrkA) and determinants for extended spectrum β-lactamase and carbapenamases (bla-IMP, bla-VIM, bla-GIM and bla-SIM) were assessed by PCR-based amplification. Results: All strains were susceptible to imipenem (p < 0.05); frequencies of resistance varied from 4% (for amikacin) and 8.7% (for meropenem) to over 30% for the other antimicrobials. About half of strains were resistant to ceftazidime, gentamicin and piperacillin. Mean MAR index was 0.31. The presence of antibiotics and nicotine at 2 and 4 mg/ml negatively affected biofilm formation for most strains. However, with amino acid starvation, almost 60% of strains retained medium or high biofilm production. Most strains harboured determinants for carbapenemase or metallo-b-lactamase, and one-third were PCR-positive for the OXA-1 gene. Strains were clustered into three groups based on ERIC-PCR analysis. Conclusion: These data suggest that certain antibiotics could inhibit biofilm production in K. pneumoniae even as multidrug resistance in this organism is evident. Further, this species has the propensity to harbour several genetic determinants for antimicrobial resistance.