Background: Kayakalpa practices, introduced under the Swachh Bharat Abhiyan, focus on improving cleanliness, hygiene, and infection control in healthcare facilities. Evaluating their effectiveness, particularly among healthcare workers (HCWs), is crucial for ensuring these objectives are met. Objective: This study aims to evaluate the awareness, adherence, and perceived effectiveness of Kayakalpa practices among healthcare workers in the South Zone PHCs of Bengaluru District, as well as to identify the factors influencing these outcomes. Methods: A cross-sectional study was conducted among 200 healthcare workers from selected PHCs in the South Zone of Bengaluru, using stratified random sampling to ensure representation across different job roles. A structured questionnaire was administered to assess awareness, adherence, perceived effectiveness, and barriers to adherence. Data were analyzed using SPSS software, employing descriptive statistics, chi-square tests for associations, correlation analysis, and multiple regression analysis to identify predictors of adherence and perceived effectiveness. Results: Awareness of Kayakalpa practices was high, with 75% of participants reporting knowledge of the guidelines. However, adherence was lower at 55%. Chi-square tests revealed significant associations between job role and adherence (χ² = 12.34, p < 0.05), and between perceived effectiveness and the frequency of training (χ² = 9.67, p < 0.01). Correlation analysis showed a significant positive relationship between awareness and adherence (r = 0.45, p < 0.01). Multiple regression analysis revealed that both awareness (β = 0.35, p < 0.05) and availability of resources (β = 0.28, p < 0.05) were significant predictors of adherence. Perceived effectiveness was significantly associated with adherence (β = 0.42, p < 0.01) and regular training (β = 0.31, p < 0.05). Discussion: The study highlights a gap between awareness and adherence to Kayakalpa practices among HCWs, with adherence being influenced by both awareness and resource availability. The effectiveness of the practices was found to be closely linked to adherence and the provision of continuous training. The chi-square analysis underscores the role of job role and training frequency in adherence and perceived effectiveness. Conclusion: While awareness of Kayakalpa practices is relatively high, adherence remains a challenge, limiting the overall effectiveness of the initiative. Targeted interventions, including regular training and improved resource allocation, are necessary to enhance adherence and maximize the impact of these practices on hygiene and infection control in PHCs.