Biofilm, a structural community of bacterial cells enclosed in a self-produced polymeric matrix which could adhere to inert or living surfaces was studied. Microorganisms that grow within the biofilm state possess several mechanisms that increase resistance to external antimicrobial treatments. The objective of the study was to determine the prevalence of biofilm forming staphylococci at the University of Ilorin Teaching Hospital. One hundred and sixty-eight staphylococcal isolates from different clinical specimens were collected in a non-repetitive manner and studied at the Microbiology Laboratory of University of Ilorin Teaching Hospital (UITH). These isolates were collected into 20% glycerol-brain heart infusion broth in vials and stored at -20oC for further processing. The isolates were re-characterized using standard microbiological techniques. Biofilm detection and quantification was carried out using modified Christensen’s microtitre plate method and the optical density determined at 450nm. The prevalence of biofilm formation among Staphylococcal isolates was 56.5%. S. aureus isolates had 52.8% while coagulase negative Staphylococci had 79.2% biofilm producers. Strong biofilm production was found to be highest in urine (35.7%), followed by wound swab (31%) and blood specimen (19.0%). Some of the wards of admission showed high prevalence of biofilm producers. Notable of the wards were General Outpatient Department (21.1%), Accident and emergency (16.8%) and Neonatal intensive care unit (13.7%). The prevalence of biofilm production at UITH is relatively high and of grave concern considering the devastating effect of antimicrobial resistance, therefore, there is a need to include biofilm detection protocol in the routine microbiological examination.