In this study, three KRN7000 analogues with variations in the sugar and glycosidic linkage were synthesised to assess their efficacy in disrupting the biofilms of S. pyogenes and P. mirabilis. All three analogues exhibited antibacterial activity, with the effects being more prominent at lower concentrations in S. pyogenes. The N-alkylated, 1-deoxy analogue emerged as the most effective, significantly reducing biofilm formation and extracellular polymeric substances (EPS) in both organisms. Microscopic analysis revealed notable disruption of biofilm structure by the analogue, resulting in a significant reduction in EPS for both organisms and decreasing cell surface hydrophobicity. These results position the KRN7000 analogue as a promising candidate for developing glycolipid-based antibiofilm agents.