Freshwater fungi Longipedicellata (Longipedicellata megafusiformis) and Wicklowia (Wicklowia fusiformispora) produced metabolites that killed pathogenic bacteria and disrupted established biofilms, the protective slime layers that make infections up to 1,000× more drug-resistant. Their extracts showed strong antibacterial activity and sharply reduced biofilm mass and metabolic activity in lab tests, indicating direct breakdown of bacterial defense structures. Metabolomic profiling revealed multiple unique antimicrobial compounds not found in current drug libraries.