Background: Peripheral intravenous catheters (PIVCs) break the skin barrier, and preinsertion antiseptic disinfection and sterile dressings are used to reduce risk of catheter-related bloodstream infection (CRBSI). In this study, the impact of PIVC skin site colonization on tip colonization and the development of CRBSI was investigated. Methods: A total of 137 patients’ PIVC skin site swabs and paired PIVC tips were collected at catheter removal, cultured, and bacterial species and clonality were identified. Results: Of 137 patients, 45 (33%) had colonized skin sites and/or PIVC tips. Of 16 patients with paired colonization of both the skin site and PIVC tips, 11 (69%) were colonized with the same bacterial species. Of these, 77% were clonally related, including 1 identical clone of Pseudomonas aeruginosa in a patient with systemic infection and the same organism identified in blood culture. Conclusions: The results demonstrate that opportunistic pathogen colonization at the skin site poses a significant risk for PIVC colonization and CRBSI. Further research is needed to improve current preinsertion antiseptic disinfection of PIVC skin site and the sterile insertion procedure to potentially reduce PIVC colonization and infection risk.