(1) Background: Gender-based violence (GBV) is widespread globally and has a myriad of adverse effects but is vastly under-reported. Health care workers are among the first responders in GBV. The objective of this study was to assess the knowledge of health workers with regard to GBV and related management guidelines and implementation. (2) Methods: The study employed a descriptive, sequential mix-method study, beginning with the quantitative part, followed by the qualitative component. Qualitative analysis was conducted using a content framework approach. (3) Results: More than two-thirds (71.79%) of health workers were found to be generally knowledgeable about gender-based violence; however, only 36.9% had good knowledge about gender-based violence management guidelines for gender-based violence and the mean value for all the items was less than 3 which indicates poor knowledge of the management guideline. Additionally, only 36.8% found the gender-based violence management guidelines useful and practical in clinical care for gender-based violence cases. (4) Conclusions: The finding of this study revealed that knowledge of gender-based violence management guideline was not adequate among health workers and rarely used during management of GBV cases. This calls for continuous training and specific refresher courses, including on-site practical sessions, professionals’ mentorship, and supervision.