: In this prospective cohort study conducted at Hijaz Hospital in Lahore from January 2022 to January 2023, we aimed to investigate the prevalence and factors influencing HCV seroconversion in hemodialysis patients. A total of 380 patients participated in the study, responding to a structured questionnaire covering sociodemographic factors and hemodialysis duration. ELISA testing was conducted quarterly, resulting in two groups: HCV-positive and HCV-negative. Results revealed that 63.4% (241 individuals) experienced seroconversion from HCV-negative to HCV-positive status during hemodialysis. Significant associations were observed between HCV seroconversion and emergency hemodialysis at a site other than the primary one (P = 0.017). Other significant associations included IV drug abuse (P = 0.007), blood transfusions (P = 0.001), and dental procedures (P = 0.005). The presence of anemia and the lack of vascular access at the start of hemodialysis were also significantly associated with HCV seroconversion (P = 0.001). However, no associations were found between HCV seroconversion and age, surgical procedures, needle stick injuries, or alcohol intake. In conclusion, this study highlights an alarming HCV seroconversion rate among hemodialysis patients. Factors such as anemia, blood transfusions, duration of dialysis, lack of vascular access, and emergency dialysis demonstrated strong correlations with HCV infectivity. These findings emphasize the importance of implementing stringent infection control measures and vigilant monitoring to mitigate the risk of HCV transmission in hemodialysis settings.