Background Needle stick injuries caused by various sharp and other items like hypodermic needlesand intravenous cannulas are important occupational hazards for healthcare workers (HCW). Preventing injuries is the most effective way to protect workers and requires good awareness and perceptions associated with practice on a daily basis. Therefore, we did a descriptive cross-sectional study involving healthcare workers in a tertiary care hospital to find the level of awareness, perception, and practice associated with needle stick injury and its prevention. Methodology A descriptive cross-sectional study was conducted in a tertiary care hospital, in south India. 400 healthcare workers (doctors, nurses, technicians, and housekeeping staff) with more than one year of experience were randomly selected. An anonymous, self-reporting, semi-structured questionnaire was administered. Results are expressed in mean, standard deviation, frequency, and percentages. Results Out of 400 respondents, 89% had good awareness about proper disposal practices. However,44% of the participants had the misbelief that recapping needles was recommended to prevent needle stick injury (NSI), and 30.5% practiced it, with the doctors being the majority. The majority (79.8%) knew that HIV, Hepatitis B & C are blood-borne pathogens that HCWs are most commonly exposed to through needle-stick injury (NSI). However, only 49% knew that Hepatitis B has the highest risk of transmission following a needle prick. 75% were aware of the correct Hepatitis B vaccination doses. Most of the healthcare workers (89.5%) claimed to be aware of the procedure and guidelines to be followed after a needle stick injury and 96% felt that they would report NSI immediately. Awareness regarding Hepatitis C prevention was comparatively poor, with only 47% having knowledge regarding the non-availability of Hepatitis C Vaccination and& 46% about the non-availability of post-exposure prophylaxis for Hepatitis C. Among the healthcare workers, 61% were worried about having needle stick injuries but 56.5% felt that their own personal safety is secondary to patient care. Among the HCWs, 91.3% believed that needle stick injuries can be prevented. Most of the participants (93.5%) ensure that others around them take extra precautions while handling sharp/ needles. The majority, i.e. 88%, utilized a designated container for disposal of sharp items, whileonly 53% of respondents utilized a needle cutter or shredder. 85% of HCWs had attended specific training programs on the usage of safe devices/sharps in the preceding one-year period and 72.8% had completed the vaccination against Hepatitis B. Conclusion Awareness regarding needle stick injury and its prevention is patchy and not adequate across different sections of healthcare workers. Perceptions regarding needle stick injury and its prevention revealed an overall positive attitude. Practices related to needle stick injury and its prevention seem to be reasonably good except when related to recapping and waiting to dispose of until the completion of the session. Training sessions need to be tailored for specific participant groups and a 'one size fits all' philosophy cannot be followed.