Objectives: This study aims to detect the presence of bacterial biofilm in the surgical operating rooms in Suez Canal University Hospital, and evaluate the activity of silver nanoparticles (AgNPs) as an antibiofilm agent and its effect in bacterial colony count in surgical operating rooms. Methods: Frequently touched surfaces in five surgical operating rooms were, swabbed with dry sterile cotton swabs such as air supply vents, beds, anesthesia devices' surfaces, doors, and walls. The bacterial samples were tested for biofilm production by microtitre plate method. Then the biofilm producers were identified using VITEK® 2 system. Silver nanoparticles were tested to inhibit the biofilm formation and to eradicate pre-formed biofilm in microtitre plate and the effect was measured spectrophotometrically. After that silver nanoparticles were tested to reduce the aerobic colony count on frequently touched surfaces besides walls and floors in operating rooms by determination the colony count before and after use of silver nanoparticles. Results: A total of 71 samples obtained from five surgical operating rooms, 19 samples were found to be strong biofilm producers and 25 were moderated biofilm producers. Silver nanoparticles and sodium hypochlorite showed no statistical differences in in vitro tests of antimicrobial activity and antibiofilm activity. Silver nanoparticles (AgNPs), in concentration 150 ppm, showed significant decrease in aerobic colony count on operating rooms frequently touched surfaces after use of AgNPs, and significant reduction of aerobic colony count (ACC) on walls and floors in comparison with sodium hypochlorite. Conclusion: Silver nanoparticles are more efficient in reduction of ACC in comparison with sodium hypochlorite.