Polybacterial infections occur when two or more distinct bacterial pathogens simultaneously contribute to the disease development and progress. Polybacterial infections are commonly observed in cystic fibrosis, chronic rhinosinusitis (CRS), dental caries, wound infections, and diabetic foot ulcers, where multiple bacteria coexist and develop biofilms to protect them from the host’s immunity and antibiotic treatments. Bacterial biofilm develops through complex interactions between bacterial communities that compete for space and nutrition. In polybacterial biofilms, diverse interactions occur considerably between bacteria, viz., mutualism, synergism, and antagonism causing various diseases in humans. Chronic infections associated with biofilms include urinary tract infections caused by Enteropathogenic Escherichia coli, chronic otitis media involving Haemophilus influenzae and Streptococcus pneumoniae, CRS by Staphylococcus aureus, and pneumonia by Pseudomonas aeruginosa. Biofilm-associated oral, ear, and wound infections are mainly polymicrobial in nature. Interactions among biofilm bacteria alter virulence, worsen inflammation, and increase antibiotic resistance, thereby complicating diagnosis and treatment. The persistence of biofilm-associated polybacterial infections coupled with antibiotic resistance underscores the need for better strategies to overcome these health complications.