Background To reduce the incidence of phlebotomy-related percutaneous injuries (PIs), factors that contribute to these injuries must be identified. This study examined institutional phlebotomy practices, policies, perceptions, and culture to identify facilitators and barriers that appear to have the greatest impact in preventing injuries. Methods During site visits at study hospitals, observational data were collected during the performance of phlebotomy. In addition, interviews and focus groups were conducted with hospital personnel involved in phlebotomy procedures. Results Nine hospitals participated in the study. A total of 126 phlebotomy procedures were observed. Health care personnel chose devices with safety features for the majority of observed procedures (n = 122, 97%). Recommended phlebotomy practices for handling needles after use were observed in 42% to 92% of procedures. Adherence varied by type of device, occupation, and facility PI rate. In the 23 interviews and 9 focus groups, participants identified factors that facilitated PI prevention such as the availability and use of devices with safety mechanisms, adherence to recommended safe needle-handling practices, and institutional phlebotomy training. Conclusion The quantitative and qualitative data indicate that a wide array of factors can affect phlebotomy-related practices and perceptions. Prevention of PIs may require comprehensive, multifaceted intervention efforts to improve the safety culture and reduce PIs and exposure to bloodborne pathogens in health care facilities.