Microbial biofilm buildup poses a significant challenge in hospital intensive care units (ICUs), by clogging healthcare devices, consequently resulting in treatment failure. The current work aimed to isolate biofilm-forming Pseudomonas aeruginosa, sequence and analyze the biofilm-forming gene (algL), and evaluate the biofilm-inhibitory potential of tomato carbon dots and manuka honey. For biofilm formation and inhibition analysis, the microtiter plate, Congo red, and tube methods were used. A total of 150 samples (swabs) from catheter mounts, ventilator masks, and urine catheters were obtained and processed in the ICUs of three hospitals in the district of Peshawar, Pakistan. P. aeruginosa was identified in 45 samples. The antibiogram of P. aeruginosa isolates demonstrated resistance to tobramycin (53.4%), ciprofloxacin (68.8%), aztreonam (42.2%), co-trimoxazole (60.6%), cefepime (40%), and ceftazidime (40%). Biofilm formation was detected in 95.5% of the samples. The biofilm was inhibited by up to 86.40% with 100 mg/mL of tomato carbon dots, whereas 68.95% inhibition was observed at 100 mg/mL of manuka honey. A total of 73.1% of P. aeruginosa isolates were positive for the presence of the algL gene. Mutational analysis revealed that the algL gene was conserved across all isolates. However, each isolated testis harbored one of the previously identified missense mutations in the algL gene. It is important to investigate the crucial factors involved in biofilm formation to develop strategies for preventing clogging of medical devices used for therapeutic purposes. Current findings may help pave the way for the development of new anti-biofilm methodologies and, in turn, effective antimicrobial therapies. Mutational analysis could yield significant insights into biofilms and inform effective inhibition strategies.