Background: Clinical audit is a core component of clinical governance and quality improvement; however, its implementation across South African public health facilities remains inconsistent. Limited facility-level evidence exists on healthcare professionals’ knowledge, perceptions, and practices regarding clinical audit, particularly in rural and resource-constrained settings. Objectives: To assess knowledge, perceptions, and practices regarding clinical audit among healthcare professionals at a district hospital in Alfred Nzo District, Eastern Cape Province, South Africa, and to identify factors associated with these domains. Methods: A descriptive cross-sectional study was conducted among healthcare professionals at Madzikane kaZulu Memorial Hospital using stratified random sampling. Of the target sample of 179 healthcare professionals, 156 participated, yielding a response rate of 87.2%. Data were collected using a structured, self-administered questionnaire adapted from a validated knowledge-attitude-practice instrument and analyzed using IBM SPSS Statistics version 30. Composite domain scores were categorized as poor (< 60%), moderate (60-79%), or good (≥80%). Associations between domain scores were assessed using Spearman’s rank-order correlation, while differences across demographic groups were examined using the Mann-Whitney U and Kruskal-Wallis tests. Results: Most participants were female (69.9%) and had >6 years of professional experience (74.4%). The median knowledge score was 72.5% (interquartile range 65.0 - 78.3), with 75.0% of participants demonstrating moderate knowledge. Although 96.1% correctly identified the purpose of clinical audit as improving patient care and outcomes, only 49.3% knew the procedural steps involved, while 37.8% incorrectly perceived clinical audit as a mechanism for disciplining staff. Perceptions were predominantly neutral (73.7%; mean score 66.1%), and only 48.7% believed that audit findings were taken seriously and acted upon by management. Clinical audit practice was moderate (mean score 57.9%), and only 39.1% of participants had received formal audit training. Knowledge was moderately and positively correlated with practice (ρ=0.537; p< 0.001). Knowledge and practice scores differed significantly across age groups (p=0.032 and p=0.004, respectively), while practice also differed significantly according to years of professional experience (p=0.021). Conclusion: Healthcare professionals at this rural district hospital demonstrated moderate knowledge and practice of clinical audit, while perceptions were predominantly neutral. Important gaps were identified in procedural knowledge, formal training, and confidence that audit findings result in management action. Strengthening structured clinical audit training, feedback mechanisms, and institutional leadership may help embed clinical audit more effectively within routine clinical governance and quality-improvement activities in resource-constrained district hospitals.