This study investigates the impact of microbial biofilms on neurosurgical site infections, focusing on prevention and treatment strategies. With 375 participants, we employed a prospective cohort design, incorporating microbiological cultures, scanning electron microscopy (SEM), and various treatment approaches to address biofilm-related infections. Our analysis revealed that biofilm formation was prevalent in 48.5% of cases, with Staphylococcus aureus and Pseudomonas aeruginosa being the most common biofilm-forming pathogens. Treatment efficacy varied, with standard antibiotics achieving a 45% success rate, biofilm-targeted antibiotics a 70% success rate, and combination therapy the highest at 80%. SEM images illustrated the progressive stages of biofilm development, highlighting the robust and resistant nature of mature biofilms. Risk factor analysis identified prolonged surgery duration, diabetes, and immunosuppression as significant contributors to biofilm-associated infections. These findings underscore the necessity for specialized treatment strategies and targeted preventive measures to enhance patient outcomes in neurosurgical infections.