Shiga Toxigenic Escherichia coli (STEC) are a group of pathogenic bacteria of high importance to food regulatory authorities, consumers, and processors due to the public health risk they pose. The objective of this research was to quantify the effect of time, temperature, and moisture on the development of STEC biofilms and subsequent contamination of beef tissues. Initially, all 13 STEC strains were screened for biofilm formation at different temperatures and growth times using crystal violet to characterize strains as weak, moderate, strong or non-biofilm forming. However, only one weak and one strong biofilm forming strain were selected for further testing. Contamination of beef was tested by growing biofilms at different temperatures (2, 10 and 25oC) and growth times (2, 4 and 6 days), followed by an aging period (2, 4, 6, 30 days) at two moisture levels (wet or dry) before testing transfer to beef tissue (lean or adipose). An additional set of coupons were additionally prepared under the same conditions and enumerated directly. The beef was centered on the coupon, followed by a 50g weight placed on the meat to simulate biofilm-to-beef transfer. The genome sequences of the two tested strains were annotated using RASTtk and screened for genes related to biofilm formation. Of the 13 tested STEC strains, strong biofilms were only detected at 25 oC after 4 days. Strain O157:H7 R5O8 formed a strong biofilm, while O26-2 only formed a weak biofilm at 25oC. Tissue type was not found to affect contamination of beef from biofilms on the stainless-steel coupons (p > 0.05). While transfer from wet biofilms to beef reached 3.2 Log (CFU/cm2) with transfer continuously decreasing over a 30-day period. Contamination from dry biofilms was minimal, as less than 1 Log (CFU/cm2) was transferred to beef or able to be enumerated directly from the coupons. Of the genes screened for only (tolA) was found to differ between O26-2 and O157:H7 R5O8. This research shows that biofilms formed by STEC can contaminate beef after prolonged periods of nutrient deprivation and desiccation. Emphasizing the need for suitable cleaning and sanitization protocols which target biofilm formation.