Background: Needle-stick injury poses a risk of transmitting pathogens such as hepatitis B, hepatitis C, and HIV, with healthcare workers being particularly vulnerable. However, it is often underreported, and there is a notable lack of knowledge, attitude, and practice studies addressing this issue in our institution. This study highlights the significance of needle-stick injury prevention and raises awareness about hepatitis B among healthcare workers. Methods: A cross-sectional study was conducted at GMC Hospital in Anantnag using convenience sampling. Data were collected from consenting healthcare workers via pre-tested questionnaires assessing knowledge, attitude, and practice related to needle-stick injury and hepatitis B. Statistical analysis was performed using Excel 2016 and IBM SPSS version 23, with Fisher's exact test (p < 0.05) used for association analysis. Results: Of 130 healthcare workers, 36.9% participants had good knowledge regarding needle-stick injury and hepatitis B, 20.8% participants demonstrated a low-to-fair attitude, and 50% participants exhibited good practices. Healthcare workers aged 40-60 had the highest rate of good practices (72.7%). Nurses (40.4%) and emergency staff (50%) displayed better knowledge, while laboratory technicians (61.1%) had lower practice scores. Among those affected by needle-stick injury, 25.4% reported an incident, with 40% occurring in surgical units. Needle-stick injury incidents were higher among female (65.6%) than male participants (34.3%), and only 36.2% of healthcare workers had completed a hepatitis B vaccination series. Conclusion: This study emphasizes the need for targeted interventions to improve needle-stick injury prevention practices and enhance vaccination completion among healthcare workers, fostering a safer healthcare environment.